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Bone formation in Transforming Growth Factor beta-1-loaded titanium fiber mesh implants
Abstract
The osteoconductive properties of porous titanium (Ti) fiber mesh with or without a calcium phosphate (Ca-P) coating and osteoinductive properties of non-coated Ti fiber mesh loaded with recombinant human Transforming Growth Factor beta-1 (rhTGF-β1) were investigated in a rabbit non-critical size cranial defect model. Nine Ca-P-coated and 18 non-coated porous titanium implants, half of them loaded with rhTGF-β1, were bilaterally placed in the cranium of 18 New Zealand White rabbits. At 8 weeks postoperative, the rabbits were sacrificed and the skulls with the implants were retrieved. Histological analysis demonstrated that in the TGF-β1-loaded implants, bone had been formed throughout the implant, up to its center, whereas in the non-loaded implants only partial ingrowth of bone was observed. Bone formation had a trabecular appearance together with bone marrow-like tissue. No difference in ingrowth could be observed between the non-TGF-β1-loaded non-coated implants and the Ca-P-coated ones. All histological findings were confirmed by image analysis: 97% ingrowth was seen in the rhTGF-β1-loaded implants, while only 57% and 54% ingrowth was observed in the non-loaded Ca-P-coated and non-coated implants, respectively. Bone surface area and bone fill were significantly higher in the rhTGF-β1-loaded implants (1.37 mm2 and 36%, respectively) than in the non-loaded implants (0.57 mm2 and 26%). No statistical difference was found for any parameter between the Ca-P-coated and non-coated implants. Quadruple fluorochrome labeling showed that in the Ti and Ti-CaP implants mainly bone guidance had occurred from the former defect edge, while in the Ti-TGF-β1 implants bone formation had mainly started in the center of a pore and proceeded in a centrifugal manner. Our results show that: (1) the combination of Ti-mesh with TGF-β1 can induce orthotopic bone formation; (2) Ti-fiber mesh has good osteoconductive properties; (3) a thin Ca-P coating, as applied in this study, does not seem to further enhance the bone-conducting properties of a titanium scaffold material.
Les propriétés ostéoconductives de mèches fibreuses en titane (Ti) poreux avec ou sans recouvrement de phosphate de calcium (Ca-P) et les propriétés ostéoinductives de la mèche fibreuse non-recouverte de titane mais chargée avec le facteur de croissance humain recombinant béta-1 (rhTGF-β1) ont étéétudiées dans un modèle de lésion crânienne chez le lapin. Neuf implants en titane recouverts de Ca-P et 18 non-recouverts, la moitié d'entre eux chargés de rhTGF-β1, ont été placés bilatéralement dans le crâne de 18 lapins blancs néo-zélandais. Huit semaines après l'opération les lapins ont été tués et les crânes ont été enlevés avec les implants. L'analyse histologique a démontré que dans les implants chargés TGF-β1, l'os s'était forméà l'intérieur de cet implant jusqu'à son centre tandis qu'au niveau des implants non-chargés seul une croissance partielle avait été observée. La formation osseuse avait une apparence trabéculaire avec du tissu ressemblant à de la moelle osseuse. Aucune différence dans la croissance n'a pûêtre observée entre les implants non-couverts et non-chargés de TGF-β1 et ceux recouverts de CaP. Toutes le découvertes histologiques ont été confirmées par l'analyse d'image: 97% de la croissance a été aperçue dans les implants chargés rhTGF-β1, tandis que seul 57% et 54% ont été respectivement observés dans les implants ni-chargés et ni-recouverts de CaP et non-recouverts. L'aire de surface osseuse et le remplissage osseux étaient significativement plus importants dans les implants chargés rhTGF-β1 respectivement (1.37 mm2 et 36%) que dans les implants non-chargés (0.57 mm2 et 26%). Aucune différence statistique n'a été trouvée pour ces paramètres entre les implants chargés et non-chargés de CaP. Un marquage quadruple au fluochrome a indiqué que dans les implants Ti et Ti-CaP la guidance osseuse se faisait par le bord de la lésion tandis que dans les implants Ti-TGF-β1, la formation osseuse avait essentiellement démarré dans le centre d'un pore et continuait ensuite de manière centrifuge. Les résultats ont montré que 1) l'association mèche-Ti avec TGF-β1 pouvait induire la formation osseuse orthotopique, 2) la mèche fibre Ti avait des bonnes propriétés ostéoconductives et 3) un fin recouvrement de CaP ne semblait pas améliorer les propriétés ostéoconductives d'un matériel en Ti.
Die osteokonduktiven Eigenschaften von porösen Titanfasergittern (Ti) mit oder ohne Kalziumphosphatbeschichtung (Ca-P) und die osteokonduktiven Eigenschaften von nichtbeschichteten Titanfasergittern, welche mit rekombiniertem humanem transformierendem Wachstumsfaktor beta-1 (rhTGF-β1) beladen waren, wurden im Kaninchenmodell mit cranialen Defekten nichtkritischer Grösse untersucht. Neun Ca-P-beschichtete und 18 nichtbeschichtete poröse Titanimplantate, von welchen die Hälfte mit rhTGF-β1 beladen waren, wurden bilateral in das Cranium von 18 weissen Neuseelandkaninchen eingesetzt. Acht Wochen postoperativ wurden die Kaninchen geopfert und die Schädel mit den Implantaten wurden entnommen. Die histologische Analyse zeigte, dass sich in den TGF-β1-beladenen Implantaten Knochen um das gesamte Implantat bis ins Zentrum gebildet hatte, während in den nichtbeladenen Implantaten nur ein partielles Einwachsen beobachtet werden konnte. Die Knochenbildung hatte ein trabekuläres Erscheinungsbild mit knochenmarkähnlichem Gewebe. Zwischen den nicht mit TGF-β1-beladenen nichtbeschichteten Implantaten und den Ca-P-beschichteten konnten keine Unterschiede im Einwachsen von Knochen beobachtet werden. Alle histologischen Resultate wurden durch Bildanalysen bestätigt: 97% Einwachsen konnte bei den rhTGF-β1-beladenen Implantaten gesehen werden, während nur 57% Einwachsen bei den nichtbeladenen Ca-P-beschichteten Implantaten und 54% Einwachsen bei den nichtbeladenen unbeschichteten Implantaten gefunden wurde. Die Oberfläche des Knochens und der Füllungsgrad mit Knochen war bei den rhTGF-β1-beladenen Implantaten (1.37 mm2 bzw. 36%) signifikant grösser als bei den nichtbeladenen Implantaten (0.57 mm2 bzw. 26%). Zwischen den Ca-P-beschichteten und den nichtbeschichteten Implantaten konnte für keinen Parameter ein statistisch signifikanter Unterschied gefunden werden. Vierfache Markierung mit Fluoroschrom zeigte, dass in bei den Ti und Ti-CaP Implantaten hauptsächlich Knochenleitung ausgehend von den früheren Defektflächen aufgetreten war, während bei den Ti-TGF-β1 Implantaten die Knochenformation v.a. in Zentrum einer Pore begonnen hatte und in zentrifugaler Art fortgeschritten war. Unsere Resultate zeigen dass: (1) die Kombination von Ti-Gitter mit TGF-β1 orthotope Knochenformation induzieren kann; (2) Ti-Fasergitter gute osteokonduktive Eigenschaften haben; (3) eine dünne Ca-P-Beschichtung, wie in dieser Studie angewendet, die knochenkonduktiven Eigenschaften eines Titangerüstmaterials nicht weiter zu verbessern scheint.
Se investigaron las propiedades osteoconductivas de una malla de fibra porosa de titanio (Ti) con o sin una cubierta de fosfato cálcico (Ca-P) y osteoinductivas de una malla de fibra de Ti sin cubierta cargada con factor tranformante recombinante humano de crecimiento beta-1 (rhTGF-β1) en un defecto de tamaño no crítico en un modelo de cráneo de conejo. Se colocaron 9 implantes de titanio poroso cubiertos de Ca-P y 18 no cubiertos, la mitad de ellos cargados con rhTGF-β1, se colocaron bilateralmente en el cráneo de 18 conejos blancos de Nueva Zelanda. Ocho semanas tras la operación se sacrificó a los conejos y se recogieron los cráneos con los implantes. El análisis histológico demostró que en los implantes cargados con rhTGF-β1 el hueso se formó a lo largo del implante hasta su centro, mientras que en los implantes no cargados solo se observó un crecimiento parcial hacia dentro. La formación de hueso tuvo una apariencia trabecular junto con tejido tipo medula ósea. No se observó diferencia entre crecimiento hacia dentro entre los implantes no cargados de rhTGF-β1 y los no cubiertos y los implantes cubiertos de Ca-P. Todos los hallazgos histológicos se confirmaron por análisis de imagen: 97% de crecimiento hacia dentro se observó en los implantes cargados de rhTGF-β1, mientras que solo el 57% y el 54% de crecimiento hacia dentro se observó en los implantes no cargados cubiertos de Ca-P y los no cubiertos respectivamente. La superficie ósea y el relleno óseo fueron significativamente mayores en los implantes cargados de rhTGF-β1 (1.37 mm2 y 36% respectivamente) que en los implantes no cargados (0.57 mm2 y 26%). No se encontró diferencia significativa para ningún parámetro entre los implantes cubiertos de Ca-P y los no cubiertos. El marcado con cuádruple fluorocromo mostró que en los implantes Ti y Ti-Cap ocurrió principalmente un guiado desde el borde del defecto inicial, mientras que los implantes Ti-rhTGF-β1 la formación de hueso empezó principalmente en el contro de un poro y continuó de una manera centrífuga. Nuestros resultados indican que: (1) la combinación de una malla de Ti con TGF-β1 puede inducir la formación ortópica de hueso; (2) la malla de fibras de Ti tiene buenas propiedades osteoconductivas; (3) una fina cubierta de Ca-P, tal como se aplica en este estudio, no parece intensificar mas las propiedades osteoconductivas de un material de soporte de titanio.
... Nachteil der passiven Adsorption ist die unkontrollierte Desorption in vivo und im sauren Milieu (Wertz et al. 1996). In vivo lösen sich fast 80% des initialen TGF- 1 -Gehalts von Titanimplantaten ab (Vehof et al. 2002). Die kovalente Bindung von Proteinen an Oberflächen verhindert unkontrollierte Desorption und kann die Stabilität gegen Biodegradation erhöhen (Müller et al. 2005). ...
... Alle bisherigen Studien bezüglich Immobilisierung von TGF- 1 basieren auf physikalischer Adsorption, die wegen des hydrophoben Charakters des Proteins sehr stark ist. Es wurde gezeigt, dass TGF- 1 seine Funktion gegenüber Osteoblasten und glatten Muskelzellen nach Adsorption an Titan und Titanlegierungen beibehält(Mann BK. et al 2001, Fischer U. et al 2003, Vehof JW. et al 2002. Der große Nachteil passiver Adsorption ist, dass unkontrollierte Desorption stattfinden kann, besonders in vivo und unter saurem pH Bedingungen(Werz O. et al 1996).Vehof et al. (2002) zeigten, dass sich in vivo fast 80% von der anfänglichen TGF- 1 Dosis von Titanfaser-Maschenimplantaten ablöste.Um die Desorption von TGF- 1 nach der Immobilisierung zu überprüfen, untersuchten wir exemplarisch, stellvertretend für alle OT, die Mediumüberstände der SuperAldehyd-OT mittels ELISA. ...
... seine Funktion gegenüber Osteoblasten und glatten Muskelzellen nach Adsorption an Titan und Titanlegierungen beibehält(Mann BK. et al 2001, Fischer U. et al 2003, Vehof JW. et al 2002. Der große Nachteil passiver Adsorption ist, dass unkontrollierte Desorption stattfinden kann, besonders in vivo und unter saurem pH Bedingungen(Werz O. et al 1996).Vehof et al. (2002) zeigten, dass sich in vivo fast 80% von der anfänglichen TGF- 1 Dosis von Titanfaser-Maschenimplantaten ablöste.Um die Desorption von TGF- 1 nach der Immobilisierung zu überprüfen, untersuchten wir exemplarisch, stellvertretend für alle OT, die Mediumüberstände der SuperAldehyd-OT mittels ELISA. Dazu wurde der Gehalt sowohl an latente ...
... Nachteil der passiven Adsorption ist die unkontrollierte Desorption in vivo und im sauren Milieu (Wertz et al. 1996). In vivo lösen sich fast 80% des initialen TGF- 1 -Gehalts von Titanimplantaten ab (Vehof et al. 2002). Die kovalente Bindung von Proteinen an Oberflächen verhindert unkontrollierte Desorption und kann die Stabilität gegen Biodegradation erhöhen (Müller et al. 2005). ...
... Alle bisherigen Studien bezüglich Immobilisierung von TGF- 1 basieren auf physikalischer Adsorption, die wegen des hydrophoben Charakters des Proteins sehr stark ist. Es wurde gezeigt, dass TGF- 1 seine Funktion gegenüber Osteoblasten und glatten Muskelzellen nach Adsorption an Titan und Titanlegierungen beibehält(Mann BK. et al 2001, Fischer U. et al 2003, Vehof JW. et al 2002. Der große Nachteil passiver Adsorption ist, dass unkontrollierte Desorption stattfinden kann, besonders in vivo und unter saurem pH Bedingungen(Werz O. et al 1996).Vehof et al. (2002) zeigten, dass sich in vivo fast 80% von der anfänglichen TGF- 1 Dosis von Titanfaser-Maschenimplantaten ablöste.Um die Desorption von TGF- 1 nach der Immobilisierung zu überprüfen, untersuchten wir exemplarisch, stellvertretend für alle OT, die Mediumüberstände der SuperAldehyd-OT mittels ELISA. ...
... seine Funktion gegenüber Osteoblasten und glatten Muskelzellen nach Adsorption an Titan und Titanlegierungen beibehält(Mann BK. et al 2001, Fischer U. et al 2003, Vehof JW. et al 2002. Der große Nachteil passiver Adsorption ist, dass unkontrollierte Desorption stattfinden kann, besonders in vivo und unter saurem pH Bedingungen(Werz O. et al 1996).Vehof et al. (2002) zeigten, dass sich in vivo fast 80% von der anfänglichen TGF- 1 Dosis von Titanfaser-Maschenimplantaten ablöste.Um die Desorption von TGF- 1 nach der Immobilisierung zu überprüfen, untersuchten wir exemplarisch, stellvertretend für alle OT, die Mediumüberstände der SuperAldehyd-OT mittels ELISA. Dazu wurde der Gehalt sowohl an latente ...
... In addition, orthopedic biomaterial surfaces have recently been functionalized to mimic various properties (such as chemistry and topography) of extracellular matrices [12]. Similarly, orthopedic devices have been used for the simultaneous loading of bioactive molecules for release at pathological sites [13]. This review is designed to highlight the evolution of biomaterials as orthopedic devices. ...
... Metal alloy-based orthopedic devices have been used for the delivery of therapeutic moieties such as drugs [45], growth factors [13] as well as bone marrow stromal cells [46], which are beyond the scope of this review. Magnesium (Mg) shows poor corrosion resistance. ...
Introduction: Titanium-based materials do not fulfill all the requirements of orthopedic implants due to a mismatch in mechanical properties with bone which are prone to change during the course of bone growth. Biofunctional biomaterials are a new class of materials that show bioactivity and adaptability at any stage of bone growth.
Areas covered: Different biofunctional biomaterials have evolved over time that can enhance calcium phosphate (CaP) precipitation, stimulate osteogenic differentiation, and can control osteoblast gene expression. These materials include metals or metal alloys, ceramics, polymers and biocomposites. Similarly, naturally-inspired nanomaterials and nanometer surface featured modified materials can enhance bone growth if created to match bone’s unique micro to nano hierarchical structure. Nanoscale manipulation of existing biomaterials can incorporate antimicrobial properties which is desirable to prevent infection and failure of orthopedic devices.
Expert commentary: Recent research trends in biofunctional biomaterials have focused to, first, understand the bone growth mechanism and, then, mimic natural bone architecture using biomaterials. Therefore, an enhanced understanding of material properties and tissue engineering principles will lead the way forward designing biofunctional biomaterials. In the future, the role of biofunctional biomaterials and orthopedic sensors will be more pronounced in terms of musculoskeletal disease prevention, diagnosis, and treatment.
... The trabecular metals exhibited higher stability and osteoconduction compared to other materials. Despite its recent evolution as biomaterial, there have been several clinical data (Zou et al. 2004;Tanzer et al. 2001;Meneghini et al. 2008;Adams et al. 2005) and preclinical studies (Vehof et al. 2000(Vehof et al. , 2002Van Den Dolder et al. 2003;Kroese-Deutman et al. 2005;Hartman et al. 2005;Habibovic et al. 2005) augmenting porous tantalum as an effective alternative for orthopaedic applications. ...
Bone defects occur due to factors such as congenital anomaly, trauma, and osseous deficiency following resection of tumours. Biomaterials are required for bone augmentation of the lost bone architecture. Clinicians attempting to regenerate the tissue and restore its function and aesthetics because of trauma, pathology, or congenital defects face a substantial challenge. The concept of using metallic foam in bone tissue engineering is a key factor in the regeneration of critical size bone defects. Significant research efforts have been dedicated to the development of metallic foams for bone tissue engineering due to their suitable mechanical and biological properties. Although, at present, most of the studies are focused on non-load bearing materials, many materials are also being investigated for hard tissue repair. Several biocompatible metallic foam materials such as titanium alloys, tantalum, iron, zinc, and magnesium alloys have been commonly employed as implants in biomedical applications. They are often used to replace and regenerate the damaged bones or to provide structural support for healing bone defects. The bone cells develop on the porous regions of the implants. These cells develop over the surface of the foam, which imparts the integrity and strength. The scaffolds help in regeneration of the biological structural components of the extracellular matrix. This chapter focuses on the commonly used metallic foams for bone tissue engineering, their properties, applications, and cellular interactions.
... TFM is composed of sintered non-woven microscale fibers of commercially pure titanium, with interconnective porosity between fibers. Over the last decades, TFM, with a fiber diameter of 50 µm and a volumetric porosity of 87%, has been extensively used in several biomedical applications, such as the soft-tissue anchorage of percutaneous devices [9,10] and a scaffold material for bone tissue engineering [11,12]. Recently, TFM with various smaller diameters, but similar porosities have been developed. ...
Objectives
This study was aimed to comparatively evaluate new bone formation into the pores of a flexible titanium fiber mesh (TFM) applied on the surface of implant.
Methods
Twenty-eight custom made cylindrical titanium implants (4 ×10 mm) with and without a layer of two different types of TFM (fiber diameter of 22 µm and 50 µm, volumetric porosity ~70%) were manufactured and installed bilaterally in the femoral condyles of 14 rabbits. The elastic modulus for these two TFM types was ~20 GPa and ~5 GPa respectively, whereas the solid titanium was ~110 GPa. The implants (Control, TFM-22, TFM-50) were retrieved after 14 weeks of healing and prepared for histological assessment. The percentage of the bone area (BA%), the bone-to-implant contact (BIC%) and amount were determined.
Results
Newly formed bone into mesh porosity was observed for all three types of implants. Histomorphometric analyses revealed significantly higher (~2.5 fold) BA% values for TFM-22 implants (30.9 ± 9.5%) compared to Control implants (12.7 ± 6.0%), whereas BA% for TMF-50 did not significantly differ compared with Control implants. Furthermore, both TFM-22 and TFM-50 implants showed significantly higher BIC% values (64.9 ± 14.0%, ~2.5 fold; 47.1 ± 14.1%, ~2 fold) compared to Control (23.6 ± 17.4%). Finally, TFM-22 implants showed more and thicker trabeculae in the peri-implant region.
Significance
This in vivo study demonstrated that implants with a flexible coating of TFM improve bone formation within the inter-fiber space and the peri-implant region.
... Although the incorporation of metal nanoparticles in polymeric scaffolds is known to effectively improve scaffold mechanical properties [65,66], the application of metal scaffolds for GF delivery is limited due to the low biodegradability, high rigidity, limited integration to the host tissue, and infection possibility of metal scaffolds [61]. Moreover, compared to polymeric scaffolds, porous metallic scaffolds mostly can only be manufactured through complex procedures, such as electron beam melting [67], layer-by-layer powder fabrication using computer-aided design strategies [68], and extrusion [69], which further limits their architecture design and application in GF delivery [61]. To avoid compromising the function and structure of new bone, the degradation rate of bone biomaterials should match the growth rate of the new structure [70]. ...
Shortcomings related to the treatment of bone diseases and consequent tissue regeneration such as transplants have been addressed to some extent by tissue engineering and regenerative medicine. Tissue engineering has promoted structures that can simulate the extracellular matrix and are capable of guiding natural bone repair using signaling molecules to promote osteoinduction and angiogenesis essential in the formation of new bone tissues. Although recent studies on developing novel growth factor delivery systems for bone repair have attracted great attention, taking into account the complexity of the extracellular matrix, scaffolding and growth factors should not be explored independently. Consequently, systems that combine both concepts have great potential to promote the effectiveness of bone regeneration methods. In this review, recent developments in bone regeneration that simultaneously consider scaffolding and growth factors are covered in detail. The main emphasis in this overview is on delivery strategies that employ polymer-based scaffolds for spatiotemporal-controlled delivery of both single and multiple growth factors in bone-regeneration approaches. From clinical applications to creating alternative structural materials, bone tissue engineering has been advancing constantly, and it is relevant to regularly update related topics.
... Similarly, TGF-β initiates and promotes heterotopic ossification in mice via recruiting mesenchymal progenitors (Wang et al., 2018). TGF-β1 induces bone closure of rabbit skull defects (Beck et al., 1993) and TGF-β1-loaded implants provoke an increased bone surface area in a rabbit cranial defect model (Vehof, Haus, de Ruijter, Spauwen, & Jansen, 2002). In contrast, TGF-β1 loaded β-TCP failed to support bone formation in a rat calvaria model (Elimelech et al., 2019) and in vitro, TGF-β reduces osteogenic differentiation in cell culture models (Noda & Rodan, 1986). ...
Acid bone lysates represent the growth factors and other molecules released during autologous graft resorption. However, the impact of these bone‐derived growth factors on the healing of bone defects has not yet been investigated. The aim of the present study was, therefore, to examine the impact of acid bone lysates adsorbed to collagen membranes on bone regeneration. To this end, in 16 female Sprague Dawley rats a standardized 5‐mm‐diameter critical size defect on the calvarial bone was created. The defects were covered with collagen membranes that had been soaked either in serum‐free media or acid bone lysates followed by lyophilization. After a healing period of four weeks, micro computed tomography (CT) and histological analyses by means of undecalcified thin ground sections were performed. Micro CT analysis of the inner 4 mm of the calvaria defect showed a greater bone defect coverage in the control group when compared to acid bone lysate group, 29.8 % (CI: 17.7 ‐ 50.3) versus 5.6 % (CI: 1.0 ‐ 29.8, p=0.03), respectively. Moreover, we found significantly more absolute bone volume in the control group when compared to acid bone lysate group, 0.59 mm³ (CI: 0.27 ‐ 1.25) versus 0.07 mm³ (CI: 0.06 ‐ 0.59, p=0.04), respectively. Histomorphometry confirmed these findings with a relative bone volume in the central compartment of 14.1% (CI: 8.4 ‐ 20.6) versus 5.6 % (CI: 3.4 ‐ 7.9, p=0.004) respectively. These findings indicate that bone‐derived growth factors contained in acid bone lysates are able to attenuate bone regeneration within collagen membranes.
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... TFM is composed of sintered non-woven microscale fibers of commercially pure titanium, with interconnective porosity between fibers. Over the last decades, TFM, with a fiber diameter of 50 µm and a volumetric porosity of 87%, has been extensively used in several biomedical applications, such as the soft-tissue anchorage of percutaneous devices [9,10] and a scaffold material for bone tissue engineering [11,12]. Recently, TFM with various smaller diameters, but similar porosities have been developed. ...
With the need of rapid healing and long-term stability of dental implants, the existing Ti-based implant materials do not meet completely the current expectation of patients. Low elastic modulus Ti-alloys have shown superior biocompatibility and can achieve comparable or even faster bone formation in vivo at the interface of bone and the implant. Porous structured Ti alloys have shown to allow rapid bone ingrowth through their open structure and to achieve anchorage with bone tissue by increasing the bone-implant interface area. In addition to the mechanical properties of implant materials, the design of the implant body can be used to optimize load transfer and affect the ultimate results of osseointegration. The aim of this narrative review is to define the mechanical properties of dental implants, summarize the relationship between implant stability and osseointegration, discuss the effect of metallic implant mechanical properties (e.g. stiffness and porosity) on the bone response based on existing in vitro and in vivo information, and analyze load transfer through mechanical properties of the implant body. This narrative review concluded that although several studies have presented the advantages of low elastic modulus or high porosity alloys and their effect on osseointegration, further in vivo studies, especially long-term observational studies are needed to justify these novel materials as a replacement for current Ti-based implant materials.
... Various surface treatments, including sintered beads, plas- ma spray, and ber mesh coating, have been applied to metal- lic biomaterials in order to improve their biocompatibility. [1][2][3][4] The roughness of the implant surface is increased by these treatments to facilitate adhesion of the body tissue to the im- plant, resulting in improved osseointegration. Many research- ers have studied cell behaviors on various metallic substrates after modiication of their surface to form nano-or mi- cro-sized grooves, gratings, dimples, and dots. ...
In this study, we evaluated the osseoconductivity of Type 316L stainless steel with self-organized nanopores of three different average diameters (26, 90, and 177 nm), formed by anodic polarization. The proliferation, alkaline phosphatase activity, and morphology of MC3T3-E1 mouse osteoblast-like cells, cultured on the self-organized nanopores were evaluated. The cell densities on samples with the nanopores were higher than those on mechanically finished surfaces that were mirror-polished or ground with #2000 SiC paper. In particular, the highest cell density and alkaline phosphatase activity were obtained on the nanoporous sample with the smallest diameter of 26 nm. Cells on the samples with 26 nm nanopores extended further and spread more filopodia compared with cells on samples with the other surface morphologies. Therefore, we concluded that self-organized nanopores with an optimal diameter (e.g., 26 nm) on Type 316L stainless steel could enhance long-term cell activity.
... The in vitro results also emphasized the role of loading concentration on the release profile [98]. Another example of obtaining desirable osteocunductive properties through incorporation of GFs in the bone implants is using the combination of a porous Ti fiber mesh and a CaP coating loaded with recombinant human Transforming GF β1 (rhTGF-β1) in a series of in vivo experiments on a rabbit non-critical size cranial defect model up to eight weeks [24,99]. ...
In addition to excellent biocompatibility and mechanical performance, the new generation of bone and craniofacial implants are expected to proactively contribute to the regeneration process and dynamically interact with the host tissue. To this end, integration and sustained delivery of therapeutic agents has become a rapidly expanding area. The incorporated active molecules can offer supplementary features including promoting oteoconduction and angiogenesis, impeding bacterial infection and modulating host body reaction. Major limitations of the current practices consist of low drug stability overtime, poor control of release profile and kinetics as well as complexity of finding clinically appropriate drug dosage. In consideration of the multifaceted cascade of bone regeneration process, this research is moving towards dual/multiple drug delivery. In this case, precise control on simultaneous or sequential delivery, considering the possible synergetic interaction of the incorporated bioactive factors is of utmost importance.
... Their biocompatibility has been extensively demonstrated, especially related to bone, both experimentally [2][3][4][5] and clinically with variable implant designs and methods of construction [6][7][8][9]. Additive manufacturing methods have been employed for a long time in fields where precise building of complex structures was necessary, such as aerospace engineering and racing cars development. ...
Additive manufacturing (AM) is a technology that enables the production of models and prosthesis directly from the 3D CAD model facilitating surgical procedures, implant quality and reducing risks. Furthermore, the additive manufacturing has been used to produce implants especially designed for a particular patient, with sizes, shapes and mechanical properties optimized, in many areas of medicine such as cranioplasty surgery. This work presents AM technologies applied to design and manufacture of a biomodel, in fact, an implant for the surgical reconstruction of a large cranial defect. A series of computed tomography data was obtained and software was used to extract the cranial geometry. The protocol presented was used for creation of anatomic biomodel of the bone defect for the surgical planning as well as to design and manufacture of the patient-specific implant, reducing duration of surgery besides improving the surgical accuracy due to preoperative planning of the anatomical details.
... TGF-β1 has been widely used to promote the osteogenesis of bone marrow stromal cells in a variety of in vitro culture systems, by supplying it in the medium continuously for >4 weeks (13)(14)(15). Arious technologies have been engineered with the aim of local delivery and controlled release of an appropriate concentration of these growth factors in vitro (16)(17)(18)(19)(20)(21)(22). Therefore, the controlled administration of TGF-β1 may represent an emerging tissue engineering technology, which may modulate cellular responses to encourage bone regeneration of skeletal defects (23,24). ...
The aim of the present study was to examine the biocompatibility of transforming growth factor-β1-silk fibroin-chitosan (TGF-β1-SF-CS) scaffolds. In order to provide an ideal scaffold for use in bone tissue engineering, TGF-β1 was introduced into the SF‑CS scaffold in order to reconstruct a three dimensional scaffold, following which hFOB1.19 osteoblast cells were seeded onto TGF‑β1‑SF‑CS and SF‑CS scaffolds. On the TGF‑β1‑SF‑CS and SF‑CS scaffolds, the cell adhesion rate increased in a time‑dependent manner. Scanning electron microscopy revealed that the cells grew actively and exhibited normal morphological features with multiple fissions, and granular and filamentous substrates were observed surrounding the cells. In addition, the cell microfilaments were closely connected with the scaffolds. The cells exhibited attached growth on the surfaces of the scaffolds, however, the growth also extended into the scaffolds. Cell Counting Kit‑8 and ALP analyses revealed that TGF‑β1 significantly promoted the growth and proliferation of the hFOB1.19 osteoblast cells in the SF‑CS scaffolds, and the enhancement of osteoblast cell proliferation and activity by TGF‑β1 occurred in a time‑dependent manner. The TGF-β1-SF-CS composite material may offer potential as an ideal scaffold material for bone tissue engineering.
... Transforming growth factor β1 (TGF-β1) has been widely used to promote chondrogenesis of BMSCs in a variety of culture systems by supplying it in the medium for the duration of culture 16,31,32 . Due to the short in vivo half-life of TGF-β isoforms and their potent action on other cell types, various technologies have been engineered with the goal of local delivery and controlled release of these growth factors in vivo [94][95][96][97][98][99] . Given the wide range of functions controlled by TGF-β1 100-102 , the goal of any such technology is to stimulate the appropriate intracellular signals to generate the desired effect on cell function. ...
The inability of articular cartilage to heal after damage or disease has motivated investigation of novel cartilage tissue engineering technologies. The objective of this thesis was to advance the use of self-assembling peptide hydrogel scaffolds for cartilage repair by encapsulating bone-marrow-stromal cells (BMSCs) and incorporating chondrogenic cues to stimulate differentiation and neotissue production. To test the hypothesis that self-assembling peptide hydrogels provide cues which enhance the chondrogenic differentiation of BMSCs, a technique for rapid, high-viability BMSC encapsulation was developed. BMSCs were cultured in two peptide hydrogel sequences and compared to agarose hydrogels. BMSCs in all three hydrogels underwent TGF-3 1-mediated chondrogenesis as demonstrated by comparable gene expression and ECM biosynthesis. Cell proliferation occurred only in the peptide hydrogels, not in agarose, resulting in higher sulfated-glycosaminoglycan content and more spatially uniform proteoglycan and type II collagen deposition. These data showed that self-assembling peptide hydrogels enhance chondrogenesis compared to agarose. To evaluate the capacity for BMSCs from young and adult equine donors to produce cartilage-like ECM, neotissue formation was compared to that for animal-matched primary chondrocytes. Young chondrocytes stimulated by TGF-PlI accumulated ECM with higher sulfated-glycosaminoglycan content than adult chondrocytes and BMSCs of either age. BMSCs produced neotissue with higher dynamic stiffness than young chondrocytes. Measurement of aggrecan core-protein and chondroitin-sulfate length by atomic-force microscopy revealed BMSCs produce longer core protein and chondroitin-sulfate, and fewer catabolic-cleavage products than chondrocytes. Therefore, BMSC-produced aggrecan appears to have a younger phenotype than chondrocyte-produced aggrecan. These advantages make BMSCs a potentially superior cell source for peptide-hydrogel-based cartilage repair. To deliver TGF-pl to BMSCs via a bioactive scaffold, BMSCs were encapsulated in peptide hydrogels with both tethered and adsorbed TGF-p1 and cultured in TGF-p 1-free medium. Chondrogenesis was compared to that of unmodified peptide hydrogels with medium-delivered TGF-p1. Adsorbed-TGF-plI peptide hydrogels stimulated chondrogenesis of BMSCs as demonstrated by cell proliferation and cartilage-like ECM accumulation, while tethered TGF-p1 was not different from TGF-pl -free controls. TGF-p1 adsorbed to self-assembling peptide hydrogels can stimulate BMSC chondrogenesis. BMSC-seeded self-assembling peptide hydrogels, modified for controlled delivery of pro-chondrogenic factors, generate cartilage-like neotissue and are compatible with a single-surgery, autologous therapy for cartilage repair.
... Examples of completely porous metal scaffolds are titanium fiber meshes with 86% porosity and a 250 mm average pore size that have been used for the ex vivo culture of rat bone marrow stromal cells under static conditions (van den Dokler et al., 2003) or in a low perfusion bioreactor (Sikavitsas et al., 2003) and sub sequent implantation in cranial defects in rats (van den Dokler et al.; Sikavitsas et al.). These scaffolds have also found application as delivery systems for transforming growth factor ß1 (TGF-b1) and have been used to repair rabbit cranial defects (Vehof et al., 2002). The main advantage of metal implants is their excellent mechanical properties, which makes them the most widely applied implant material used in bone surgical repairs. ...
... It has been repeatedly suggested [11][12][13][14][15][16] that making prostheses of porous metal might help to solve both the poor bonding and the stress shielding problems. Provided the pore size is suitable (~100-300 µm) [17] and the internal surfaces are bio-compatible or bio-active, bone in-growth can readily occur, leading to strong interfacial adhesion. ...
This work relates to porous material made by bonding together fibres of a magnetic material. When subjected to a magnetic field, the array deforms, with individual fibres becoming magnetised along their length and then tending to line up locally with the direction of the field. An investigation is presented into the concept that this deformation could induce beneficial strains in bone tissue network in the early stages of growth as it grows into the porous fibre array. An analytical model has been developed, based on the deflection of individual fibre segments (between joints) experiencing bending moments as a result of the induced magnetic dipole. The model has been validated via measurements made on simple fibre assemblies and random fibre arrays. Work has also been done on the deformation characteristics of random fibre arrays with a matrix filling the inter-fibre space. This has the effect of reducing the fibre deflections. The extent of this reduction, and an estimate of the maximum strains induced in the space-filling material, can be obtained using a simple force balance approach. Predictions indicate that in-growing bone tissue, with a stiffness of around 0.01-0.1 GPa, could be strained to beneficial levels (~1 millistrain), using magnetic field strengths in current diagnostic use (~1 Tesla), provided the fibre segment aspect ratio is at least about 10. Such material has a low Young's modulus, but the overall stiffness of a prosthesis could be matched to that of cortical bone by using an integrated design involving a porous magneto-active layer bonded to a dense non-magnetic core.
... Furthermore, it is opposite to bone formation in other scaffold materials like titanium fiber mesh, which is reported to start in the center of the pore. 39,40 Finally, histomorphometrical analysis included quantification of the cement surface as indicator of macroporosity and implant degradation. Degradation of the cement was not observed in this study. ...
... Metal foams can be produced either by gas entrapment [11] or by using a temporary, solid filling phase prior to solidification [12]. Deposition through polystyrene latex beads [13], plasma-spraying [14] onto a solid sore and titanium wire into meshing via sintering [15] are other alternatives. Each of these methods have their own strengths but all have weaknesses. ...
The potential of engineered porous materials for aiding the osseointegration of metallic dental and orthopedic implants is well established. A range of techniques, such as powder metallurgy, plasma-spraying and titanium fiber meshing have been tried to produce the implant structures with control on porosity and other pertinent characteristics. In the present work, direct laser deposition of pure titanium powder (particle size: -100/+63 microns) was carried out to form porous structure. A 1 kW fiber laser, integrated with lateral powder feed nozzle and workstation, was used at different primary process parameters (laser power, powder mass flow rate, transverse speed, hatch parameter) to fabricate a number of multi-layer structures. The relevant structural parameters of these fabricated structures were studied as a function of the process parameters using various techniques. The result of these studies are presented.
... 34 There are likely multiple mechanisms driving TGF-b1 adsorption including electrostatic interactions associated with the peptide hydrogel polyampholytic composition as well as excluded volume effects associated with both peptide and agarose. TGF-b1 has been adsorbed to a wide range of materials including titanium fiber, 35 collagen-coated and uncoated titanium alloys, 36 acidic gelatin, 9,10 and functionalized dextrose. 13 The capacity for TGF-b1 to adsorb to this diverse range of materials is consistent with our results showing TGF-b1 adsorption to both uncharged agarose hydrogels and to an even greater extent to amphiphilic, zwitter-ionic peptide hydrogels. ...
Tissue engineering strategies for cartilage defect repair require technology for local targeted delivery of chondrogenic and anti-inflammatory factors. The objective of this study was to determine the release kinetics of transforming growth factor β1 (TGF-β1) from self-assembling peptide hydrogels, a candidate scaffold for cell transplant therapies, and stimulate chondrogenesis of encapsulated young equine bone marrow stromal cells (BMSCs). Although both peptide and agarose hydrogels retained TGF-β1, 5-fold higher retention was found in peptide. Excess unlabeled TGF-β1 minimally displaced retained radiolabeled TGF-β1, demonstrating biologically relevant loading capacity for peptide hydrogels. The initial release from acellular peptide hydrogels was nearly 3-fold lower than agarose hydrogels, at 18% of loaded TGF-β1 through 3 days as compared to 48% for agarose. At day 21, cumulative release of TGF-β1 was 32-44% from acellular peptide hydrogels, but was 62% from peptide hydrogels with encapsulated BMSCs, likely due to cell-mediated TGF-β1 degradation and release of small labeled species. TGF-β1 loaded peptide hydrogels stimulated chondrogenesis of young equine BMSCs, a relevant preclinical model for treating injuries in young human cohorts. Self-assembling peptide hydrogels can be used to deliver chondrogenic factors to encapsulated cells making them a promising technology for in vivo, cell-based regenerative medicine.
... [31][32][33][34] The methods that have been used for immobilization of peptides to surfaces include use of different substances such as CaP-, chondroitin sulphate-, collagen-, and fibrinogen coatings. 32,33,[35][36][37][38][39][40] The later film model is similar to the one used in the present study. In this early proof of concept, the intention was not to deliver a specific concentration but rather to establish a fully covered titanium surface with insulin. ...
The possibility to control bone formation would be favorable in many areas of medicine, where bone defects is still a major challenge. Insulin has been suggested to exert both systemic and local anabolic effects in bone tissues. This raised the question whether locally administrated insulin could provide new therapeutic strategies for patients with local bone defects and impaired bone healing. The aim of this study was to evaluate bone formation in non-diabetic rats when local insulin is administered. This study differs from previous reports in two aspects: the use of non-diabetic animals and locally administered insulin. Twenty-four implants were inserted into 12 rats-one insulin-coated and one control-in each tibia for four weeks. Interferometry and histomorphometry were used to evaluate the surface topography and bone formation, respectively. Results demonstrated no significant changes in surface topography after insulin immobilization. Histomorphometry revealed significantly more bone around the insulin-coated implants (BA) (p = 0.005) and a similar amount of bone at the implant surface (BIC) (p = 0.117) compared with the controls. It was concluded that locally administered insulin from a titanium implant surface has the potential to increase bone formation not only in diabetic subjects but also in non-diabetic subjects. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2012.
Peri-implant disease (PID) is a general term for inflammatory diseases of soft and hard tissues that occur around implants, including peri-implant mucositis and peri-implantitis. Cytokines are a class of small molecule proteins, which have various functions such as regulating innate immunity, adaptive immunity, and repairing damaged tissues. In order to explore the characteristics and clinical significance of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and tumor growth factor (TGF)-β1 expression levels in serum of patients with peri-implant disease, 31 patients with PID and 31 patients without PID were enrolled. The modified plaque index (mPLI), modified sulcus bleeding index (mSBI), and peri-implant probing depth (PD) were recorded. The levels of serum TNF-α, IL-6, IL-10, and TGF-β1 were detected by ELISA. TNF-α, mPLI, mSBI, and PD levels were significantly higher in the PID group. TGF-β1 levels were significantly higher in the control group. There was a significant positive correlation between TNF-α and mPLI, mSBI, and PD. TGF-β1 was negatively associated with TNF-α, mPLI, mSBI, and PD. Multiple logistic regression analysis showed that TNF-α and PD were risk factors for the severity of PID. The receiver operating curve analysis showed that high TNF-α levels (cut-off value of 140 pg/mL) and greater PD values (cut-off value of 4 mm) were good predictors of PID severity with an area under the curve of 0.922. These results indicated that TNF-α and PD can be used as a biological indicator for diagnosing the occurrence and progression of PID.
Particulate autologous tooth roots are increasingly used for alveolar bone augmentation; however, the proteomic profile of acid dentin lysate and the respective cellular response have not been investigated. Here we show that TGF-β1 is among the 226 proteins of acid dentin lysate (ADL) prepared from porcine teeth. RNA sequencing identified 231 strongly regulated genes when gingival fibroblasts were exposed to ADL. Out of these genes, about one third required activation of the TGF-β receptor type I kinase including interleukin 11 (IL11) and NADPH oxidase 4 (NOX4). Reverse transcription-quantitative polymerase chain reaction and immunoassay confirmed the TGF-β-dependent expression of IL11 and NOX4. The activation of canonical TGF-β signaling by ADL was further confirmed by the phosphorylation of Smad3 and translocation of Smad2/3, using Western blot and immunofluorescence staining, respectively. Finally, we showed that TGF-β activity released from dentin by acid lysis adsorbs to titanium and collagen membranes. These findings suggest that dentin particles are a rich source of TGF-β causing a major response of gingival fibroblasts.
Background
Transforming growth factor‐β (TGF‐β1) enhances mesenchymal stem cell (MSC) differentiation into osteoblasts.
Purpose
The aim of the study was to assess whether TGF‐β1 loaded onto β‐tricalcium phosphate (β‐TCP) synthetic scaffold enhances bone regeneration in a rat calvaria model. The release kinetics of TGF‐β1 from β‐TCP scaffold was evaluated in vitro.
Materials and Methods
TGF‐β1 in various concentrations (1‐40 ng/mL) was loaded onto the β‐TCP scaffold, and release kinetics was monitored by ELISA. The effect of TGF‐β1 on the proliferation of MSCs was assessed using AlamarBlue, and MSC differentiation was evaluated by Alizarin Red quantification assay.Bone augmentation following transplantation of TGF‐β1 loaded onto β‐TCP in a rat calvaria model was evaluated in vivo.
Results
Greater TGF‐β1 release from the 40 ng/mL concentration was found. A suppressive effect of TGF‐β on the MSCs proliferation was observed with maximum inhibition obtained with 40 ng/mL compared to the control group (P = .028). A positive effect on MSCs osteogenic differentiation was found.Bone height and bone area fraction in vivo were similar with or without TGF‐β1; however, blood vessel density and degradation of the scaffold were significantly higher in the TGF‐β1 group.
Conclusion
TGF‐β1 adsorbed to β‐TCP stimulated angiogenesis and scaffold degradation that may enhance bone formation.
Additive Manufacturing (AM) as an engineering technology has enabled several advances in the medical field, particularly as far as surgical planning is concerned. The use of AM in medicine has added, in an era in which so many new technologies are constantly being developed, the possibility of carrying out surgical planning and simulation using a three-dimensional physical model that is realistic and very true to the patient’s own anatomy. AM is a technology that enables the production of physical object or anatomic models – called “biomodels” – directly from a 3D virtual model (obtained by computed tomography or magnetic resonance imaging techniques) from microscopic powder. This article presents AM technologies applied to the design and manufacture of a biomodel, in this case, an implant for the surgical reconstruction of large cranial defects. The protocol presented was used to create an anatomic biomodel of the bone defect for the surgical planning stage, and the design and manufacture of the patient-specific implant, for the actual surgery, reducing the duration of the surgery in addition to improving the surgical accuracy due to preoperative planning of the anatomical details.
Tissue engineering is intended to manipulate living cells to help develop substitutes for native tissues or remodel tissue. Bioartificial tissues are commonly explored in various tissue engineering ventures to overcome the disadvantages of working with native tissue. Poly(propylene fumarate) is a potential biomaterial for bioartificial bone grafts. The polymer’s many desirable physical and chemical properties have drawn significant research interest. This miniature review is intended to cover a small portion of the investigations of poly(propylene fumarate) as a bone tissue engineering biomaterial.
Tissue engineering has emerged as a response to the problems associated with the substitution of tissues lost due to diseases or trauma. Nowadays, xenografts, allografts or autografts are commonly used to replace the damaged tissue. However, these options involve numerous drawbacks such as rejection, chronic inflammation and severe organ donor shortages. Tissue engineering keeps the promise to overcome these limitations by creating biological substitutes capable of replacing the injured tissue. Cells, temporary 3D constructs and biochemical signals that trigger tissue regeneration cascades are the three major components for engineering tissues. The adequate combination of these components leads to the successful repair of damaged tissues. In addition to these three main elements, the use and development of bioreactors has come out as an essential tool for the study and achievement of engineered tissues under in vitro conditions while mimicking the in vivo environment. Thus, tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences to develop biological substitutes that restore, maintain, or improve the tissue functions. This chapter discusses the challenge of creating engineered bone tissues by combining biological and engineering skills. In particular, it will review the issue of stem cell biology, the fabrication methods and artificial materials used for 3D constructs, the principal growth factors used in bone applications and the key role played by bioreactors. More importantly, it will highlight the interdependence of all these parameters and the need for interdisciplinary research for a successful approach.
There are different approaches for treatment of bone fractures; one of them is bone tissue engineering. There are great varieties of clinical cases when the bone grafts are needed. All of them implies to fill a relevant big gap of bone with bone tissue or to supply the bone with a good bioconductive material. One of the most successful approaches for treatment of such defects is bone tissue engineering, which implies cells and scaffold interactions. The most promising cells are mesenchymal stem cells. They fill a porous scaffold and are cultivated in vitro, after that this construct is transplanted into the defect. There are different approaches in creation of bone tissue transplant. All of them means to use different materials for scaffolds (bioceramics, bioglass, demineralized bone matrix etc.), with different properties, different cells (differentiated or stem cells), and different methods of stem cell placement and retention in those materials - it implies different number of cells, different methods of loading those cells, usage of some factors that helps for bone formation and blood vessels invasion. Here we would like to make a review of different approaches in bone tissue engineering and to tell what was done in our laboratory in this area.
Orthopedic and dental implants replace millions of arthritic, traumatic or lost skeletal or dental structures. All orthopedic or dental implants can fail, and the central reason for failure is that metallic implants do not remodel with host tissue. Current implants rely primarily on tissue growth onto implants or an 'outside-in' strategy. This chapter discusses an 'inside-out' strategy to induce tissue ingrowth by cytokine delivery. Drug-eluting porous implants have the advantage not only of reducing bulk metal mass, but also of harboring cytokines that are programmed to release into surrounding tissue. This coupled inside-out and outside-in strategy improves bone ingrowth.
Purpose:
To investigate the osseointegration properties of prototyped implants with tridimensionally interconnected pores made of the Ti6Al4V alloy and the influence of a thin calcium phosphate coating.
Methods:
Bilateral critical size calvarial defects were created in thirty Wistar rats and filled with coated and uncoated implants in a randomized fashion. The animals were kept for 15, 45 and 90 days. Implant mechanical integration was evaluated with a push-out test. Bone-implant interface was analyzed using scanning electron microscopy.
Results:
The maximum force to produce initial displacement of the implants increased during the study period, reaching values around 100N for both types of implants. Intimate contact between bone and implant was present, with progressive bone growth into the pores. No significant differences were seen between coated and uncoated implants.
Conclusion:
Adequate osseointegration can be achieved in calvarial reconstructions using prototyped Ti6Al4V Implants with the described characteristics of surface and porosity.
Drug delivery through tissue-engineered scaffolds provides a composite approach to address the regenerative limitations of simple material implantation, providing expanded avenues for therapeutic tissue-repair strategies in the clinic. Both nano- and microfibrous scaffolds generated by a variety of techniques have been investigated for their potential in drug-delivery applications. While nanofibers mimic the structure and organization of natural extracellular matrix, microfibers provide more sustained release of drugs, larger pores to facilitate cell infiltration, and improved mechanical support. Various methods exist to embed drugs within the fiber matrix to modulate the release kinetics specific to the tissue-engineering application. The current article reviews the established and emerging fabrication methods for drug-loaded fiber-based scaffolds and addresses how further combination into composite scaffolds can enhance drug delivery and tissue regeneration.
In orthopaedic and dental implantology, novel tools and techniques are being sought to improve the regeneration of bone tissue. Numerous attempts have been made to enhance the osteoconductivity of titanium prostheses, including modifications in their surface properties and coating with layers of calcium phosphate. The technique whereby such layers are produced has recently undergone a revolutionary change, which has had profound consequences for their potential to serve as drug-carrier systems. Hitherto, calcium phosphate layers were deposited upon the surfaces of metal implants under highly unphysiological physical conditions, which precluded the incorporation of proteinaceous osteoinductive drugs. These agents could only be adsorbed, superficially, upon preformed layers. Such superficially adsorbed molecules are released too rapidly within a biological milieu to be effective in their osteoinductive capacity. Now, it is possible to deposit calcium phosphate layers under physiological conditions of temperature and pH by the so-called biomimetic process, during which bioactive agents can be coprecipitated. Since these molecules are integrated into the inorganic latticework, they are released gradually in vivo as the layer undergoes degradation. This feature enhances the capacity of these coatings to act as a carrier system for osteogenic agents.
Replacing malfunctioning tissues with titanium-based implants has become a widespread practice spurred by population ageing. Advances in biomaterials, technology and implantation protocols have led to increasing expectations on the applicability and durability of implants. The field has recently moved from a bioinert to a bioactive paradigm due to surface modifications that trigger specific responses on the surrounding tissues. Biopolymeric surface coatings have taken up a central role in these developments. The use of these and other biomimetic strategies on implants provides greater control over material-cell interactions and it is aimed at improving long-term clinical results by replicating some of the structures and mechanisms of living tissues. This review summarizes the state of the art of biomimetic implants and discusses the main directions and challenges of this field towards a more predictable and successful implant osseointegration.
1. Background Replacement of hip, knee and other joints, usually as a treatment for degenerative arthritis, is becoming increasingly common, with the worldwide market currently worth about $5 billion and an estimated annual growth rate of around 9%. These operations bring pain relief to millions, but the treatment is plagued by a substantial problem. The stem of the prosthesis, which is commonly pushed down into a recess in the host bone, often becomes loose after a time. The problem is getting worse as joint replacement rates rise and operations are carried out on younger and more active patients. Prosthetic implants are attached to bone either with cement or via bone in-growth into a rough or porous surface. Although bone cement provides immediate fixation, cemented implants frequently loosen in time due to the poor wear and fatigue properties of such cement. Furthermore, in-vivo polymerization is likely to take place, with deleterious effects on the surrounding tissue. Strong bone-implant bonding can be achieved in the absence of cement by bone tissue growth into an implant surface which is rough or porous, preferably with channels of around 100-300 µm in diameter (Bobyn, Pilliar et al. 1980). However, this does not occur very readily or quickly and might typically take at least a couple of weeks - a period during which there is a serious danger of complete debonding if exercise is undertaken prematurely. It is now well established (Frost 1987; Akhouayri, Lafage-Proust et al. 2000; Mosley 2000) that bone growth is stimulated by mechanical stress and becomes sluggish in its absence. Resultant phenomena include loss of bone density and strength in astronauts after extended periods in a hypo- gravity environment and localised bone resorption adjacent to prosthetic implants, as a consequence of stress shielding. This latter effect arises because prostheses are stiffer than surrounding bone, inhibiting it from being strained. (Most metals have a stiffness of about 100-200 GPa, whereas that of cortical bone is about 7-27 GPa).
An recent innovation in medicine is the application of the biomaterials in bone healing process. Thus, this work has the objective to present an overview of the uses and insertions of these biomaterials and its components mostly used in the bone repair.
Since many natural and biological materials are cellular, often with relatively high porosity levels (although sometimes these pores are partly filled with a fluid or a soft and compliant solid in the natural state), various types of porous materials are of interest for biological applications. A key feature for such applications is the space afforded for invasion, first by cells and ultimately by osseous tissue and vasculature. The surface should be chemically and topologically suitable for cells to penetrate and interlock. There is evidence that fine scale topographic features can affect both the adhesion and ingrowth of cells. One way of creating topographic features, such as terraces, is to employ suitable heat treatments so as to expose preferentially the low surface energy crystallographic planes via surface diffusion. The topography and crystallography of surface terraces, generated on solid-state-sintered ferritic stainless-steel fibre networks, have been characterised by electron back-scattered diffraction, atomic force microscopy and scanning electron microscopy. Initial work on the effect of these fine scale topographic features on cell proliferation shows encouraging results.
Two types of entangled titanium wire materials (ETWMs) have been fabricated through different procedures. Their entangled structure, compressive mechanical properties, and strain hysteresis behavior have been investigated. The experiment has demonstrated that the entangled wire structure can be adjusted when different fabrication methods are applied. The entangled wire morphologies can be normal entangled wires or coiled wires, and the pore size distributions are alternative so as to adapt the entangled wire morphology. The ETWMs with normal wire exhibit 2.6±0.1 to 31.1±0.8MPa yield strength and 135.3±2.9 to 816.5±8.4MPa Young's modulus in the range of 77.6±0.2% to 47.8±0.4% porosity. While the ETWMs with coiled wire exhibit 1.1±0.1 to 19.1±0.5MPa yield strength and 27.4±0.5 to 623.2±5.8MPa Young's modulus in the same porosity range. The two types of the ETWMs reveal significant strain hysteresis effect, which becomes stronger as the porosity increases. The coiled wire in the entangled wire structure improves the hysteresis effect. Such pseudo-elastic hysteresis behavior and the damping and energy dissipation capability are very promising for applications in engineering, biomedicine, aviation, astronavigation, and other industries.
This eight-chapter monograph intends to present basic principles and applications of biomechanics in bone tissue engineering in order to assist tissue engineers in design and use of tissue-engineered products for repair and replacement of damaged/deformed bone tissues. Briefly, Chapter 1 gives an overall review of biomechanics in the field of bone tissue engineering. Chapter 2 provides detailed information regarding the composition and architecture of bone. Chapter 3 discusses the current methodologies for mechanical testing of bone properties (i.e., elastic, plastic, damage/fracture, viscoelastic/viscoplastic properties). Chapter 4 presents the current understanding of the mechanical behavior of bone and the associated underlying mechanisms. Chapter 5 discusses the structure and properties of scaffolds currently used for bone tissue engineering applications. Chapter 6 gives a brief discussion of current mechanical and structural tests of repair/tissue engineered bone tissues. Chapter 7 summarizes the properties of repair/tissue engineered bone tissues currently attained. Finally, Chapter 8 discusses the current issues regarding biomechanics in the area of bone tissue engineering.
Table of Contents: Introduction / Bone Composition and Structure / Current Mechanical Test Methodologies / Mechanical Behavior of Bone / Structure and Properties of Scaffolds for Bone Tissue Regeneration / Mechanical and Structural Evaluation of Repair/Tissue Engineered Bone / Mechanical and Structural Properties of Tissues Engineered/Repair Bone / Current Issues of Biomechanics in Bone Tissue Engineering
ABSTRACT The use of rabbits for experimental research has a long historical tradition. The aim of this review consists in outlining the use of the rabbit for research in implant dentistry and related tissue regeneration. Rabbits appear as a first-hand choice for fundamental implant design studies because of their size, easy handling, short life span, and economical aspects in purchasing and sustaining. In the following, the various anatomical sites in the rabbit will be summarized to provide an overview of current possibilities and limitations of this model for bone research in oral implantology.
Reconstruction and regeneration of new tissues are challenges facing scientists, technologists and clinicians. This review describes strategies of selection and design of biomaterials having significant impact on various possible synthesis routes for scaffold fabrication. The criteria for three-dimensional (3D) scaffold architectures are explored in tandem with biomaterial properties such as porosity, interconnectivity and mechanical integrity. The cell-surface biointerface is outlined in terms of biomaterial composition, target tissues and biological evaluation with emphasis on bone tissue engineering. Comparative merits and demerits of conventional and rapid prototyping (RP) approaches of fabrication are discussed. The conventional methods are often simple to design, inexpensive and flexible to optimise or modulate physicochemical properties. Despite being expensive and suffering from certain drawbacks of choice of materials and capital costs many generic RP techniques are extremely attractive in their ability to mimic new tissue structures and possibility of incorporating pharmaceutical agents. The future directions include scaffold development using nanobiomaterial based biosystems /biointerfaces where cell biology including genetically modified tissue engineering approaches can play a cross-disciplinary role for the success of tissue augmentation.
The grafting of bone in skeletal reconstruction has become a common task of the orthopedic surgeon. The need for reconstruction
or replacement is often the result of trauma, congenital malformations, or cancer. Reconstructive surgery is based upon the
principle of replacing defective tissue with viable, functioning alternatives. Various materials have been used to treat the
defects, including autogenous bone and alloplastic materials. Grafting materials are necessary to bridge defects or to increase
the bone volume. At present, autologous bone is the gold standard, but it has important disadvantages, including donor-site
morbidity, limited availability, and unpredictable resorption characteristics. These factors have stimulated the search for
other materials that can replace autogenous bone.
Mature adipocyte-derived dedifferentiated fat (DFAT) cells rapidly differentiate into osteoblasts under three-dimensional culture conditions. However, it has not been demonstrated that DFAT cells can differentiate into osteoblasts in a rigid scaffold consisting of titanium fiber mesh (TFM). We examined the proliferation and osteogenic differentiation ability of DFAT cells using TFM as a scaffold. DFAT cells derived from rabbit subcutaneous fat were seeded into TFM and cultured in osteogenic medium containing dexamethasone, L: -ascorbic acid 2-phosphate and β-glycerophosphate for 14 days. In scanning electron microscopy (SEM) analysis, well-spread cells covered the titanium fibers on day 3, and appeared to increase in number from day 3 to 7. Numerous globular accretions were found and almost completely covered the fibers on day 14. Cell proliferation, as measured by DNA content in the TFM, was significantly higher on day 7 compared with that of day 1. Osteocalcin and calcium content in the TFM were significantly higher on day 14 compared to those of days 1, 3, and 7, indicating DFAT cells differentiated into osteoblasts. We theorize that globular accretions observed in SEM analysis may be calcified matrix resulting from osteocalcin secreted by osteoblasts binding calcium contained in fetal bovine serum. In this study, we demonstrated that DFAT cells differentiate into osteoblasts and deposit mineralized matrices in TFM. Therefore, the combination of DFAT cells and TFM may be an attractive option for bone tissue engineering.
A kind of porous metal-entangled titanium wire material has been investigated in terms of the pore structure (size and distribution), the strength, the elastic modulus, and the mechanical behavior under uniaxial tensile loading. Its functions and potentials for surgical application have been explained. In particular, its advantages over competitors (e.g., conventional porous titanium) have been reviewed. In the study, a group of entangled titanium wire materials with non-woven structure were fabricated by using 12-180 MPa forming pressure, which have porosity in a range of 48%-82%. The pores in the materials are irregular in shape, which have a nearly half-normal distribution in size range. The yield strength, ultimate tensile strength, and elastic modulus are 75 MPa, 108 MPa, and 1.05 GPa, respectively, when its porosity is 44.7%. The mechanical properties decrease significantly as the porosity increases. When the porosity is 57.9%, these values become 24 MPa, 47.5 MPa, and 0.33 GPa, respectively. The low elastic modulus is due to the structural flexibility of the entangled titanium wire materials. For practical reference, a group of detailed data of the porous structure and the mechanical properties are reported. This kind of material is very promising for implant applications because of their very good toughness, perfect flexibility, high strength, adequate elastic modulus, and low cost.
We have investigated the ability of exogenous transforming growth factor-beta (TGF-beta) to induce osteogenesis and chondrogenesis, critical events in both bone formation and fracture healing. Daily injections of TGF-beta 1 or 2 into the subperiosteal region of newborn rat femurs resulted in localized intramembranous bone formation and chondrogenesis. After cessation of the injections, endochondral ossification occurred, resulting in replacement of cartilage with bone. Gene expression of type II collagen and immunolocalization of types I and II collagen were detected within the TGF-beta-induced cartilage and bone. Moreover, injection of TGF-beta 2 stimulated synthesis of TGF-beta 1 in chondrocytes and osteoblasts within the newly induced bone and cartilage, suggesting positive autoregulation of TGF-beta. TGF-beta 2 was more active in vivo than TGF-beta 1, stimulating formation of a mass that was on the average 375% larger at a comparable dose (p less than 0.001). With either TGF-beta isoform, the dose of the growth factor determined which type of tissue formed, so that the ratio of cartilage formation to intramembranous bone formation decreased as the dose was lowered. For TGF-beta 1, reducing the daily dose from 200 to 20 ng decreased the cartilage/intramembranous bone formation ratio from 3.57 to zero (p less than 0.001). With TGF-beta 2, the same dose change decreased the ratio from 3.71 to 0.28 (p less than 0.001). These data demonstrate that mesenchymal precursor cells in the periosteum are stimulated by TGF-beta to proliferate and differentiate, as occurs in embryologic bone formation and early fracture healing.
Bone growth into cementless prosthetic components is compromised by osteoporosis, by any gap between the implant and the bone, by micromotion, and after the revision of failed prostheses. Recombinant human transforming growth factor-β1 (rhTGF-β1) has recently been shown to be a potent stimulator of bone healing and bone formation in various models in vivo.
We have investigated the potential of rhTGF-β1, adsorbed on to weight-loaded tricalcium phosphate (TCP) coated implants, to enhance bone ongrowth and mechanical fixation. We inserted cylindrical grit-blasted titanium alloy implants bilaterally into the weight-bearing part of the medial femoral condyles of ten skeletally mature dogs. The implants were mounted on special devices which ensured stable weight-loading during each gait cycle. All implants were initially surrounded by a 0.75 mm gap and were coated with TCP ceramic.
Each animal received two implants, one with 0.3 μg rhTGF-β1 adsorbed on the ceramic surface and the other without growth factor. Histological analysis showed that bone ongrowth was significantly increased from 22 ± 5.6% bone-implant contact in the control group to 36 ± 2.9% in the rhTGF-β stimulated group, an increase of 59%. The volume of bone in the gap was increased by 16% in rhTGF-β1-stimulated TCP-coated implants, but this difference was not significant. Mechanical push-out tests showed no difference in fixation of the implant between the two groups. Our study suggests that rhTGF-β1 adsorbed on TCP-ceramic-coated implants can enhance bone ongrowth.
Several lines of evidence suggest that local production of transforming growth factor-beta (TGF-beta) contributes to renal disease, particularly to the accumulation of the extracellular matrix protein that characterizes glomerulosclerosis and interstitial fibrosis. We have examined whether elevated levels of circulating TGF-beta adversely affect the kidney. We have studied mice that are transgenic for an active form of TGF-beta 1 under the control of murine albumin promoter and enhancer DNA sequences. These mice express the transgene exclusively in the liver and have elevated plasma concentrations of TGF-beta 1. Renal disease was seen in two of three lines of Alb/TGF-beta 1 transgenic mice; these two lines had the highest levels of hepatic transgene expression and the highest plasma TGF-beta 1 levels. Histologic abnormalities, which included mesangial expansion and thickened capillary loops, were noted in the glomeruli by 3 weeks of age. Interstitial fibrosis and tubular atrophy appeared subsequently. Mice from Line 25, the line with highest levels of TGF-beta 1, developed proteinuria by 5 weeks of age. These mice subsequently manifested nephrotic syndrome with ascites and progressive azotemia; uremic death occurred in more than 25% of the mice by 15 weeks of age. The glomeruli contained immune deposits in subendothelial and mesangial locations, but complement deposition was infrequent. Ultrastructural examination revealed an increase in extracellular matrix material, including collagen fibrils, in subendothelial and mesangial locations. Increased levels of circulating TGF-beta 1 induced progressive renal disease that was characterized by mesangial expansion, accumulation of glomerular immune deposits and matrix proteins, and interstitial fibrosis in this transgenic mouse model. These data suggest that chronically elevated circulating levels of TGF-beta 1 induce progressive glomerulosclerosis.
Transforming growth factor-beta 1 (TGF-beta 1) is a polyfunctional regulatory cytokine that has been shown to have roles in extracellular matrix interactions, soft tissue healing, and osteogenesis. This study was undertaken to determine the efficacy of TGF-beta 1 in the formation of functionally normal bone in tibial-diaphyseal defects.
Seven hundred fifty micrograms of recombinant human TGF-beta 1 was added to a guanidine-extracted demineralised bone matrix (Gu-DBM) carrier and the implants were used to fill a 2.5 cm tibial diaphyseal defect in skeletally mature female sheep. The defects were allowed to heal over a 12-week period. After sacrifice, they were analyzed using four-point bending mechanical testing.
Implants with TGF-beta 1 showed complete bony bridging of the defect and stress-strain curves similar to the normal contralateral limb, while implants with the carrier alone failed to bridge the gap.
These results demonstrate the ability of TGF-beta 1 to induce new bone which has structural and functional characteristics similar to normal bone.
Transgenic mice overexpressing a constitutively active human TGF-beta1 under control of the rat phosphoenolpyruvate carboxykinase regulatory sequences developed fibrosis of the liver, kidney, and adipose tissue, and exhibited a severe reduction in body fat. Expression of the transgene in hepatocytes resulted in increased collagen deposition, altered lobular organization, increased hepatocyte turnover, and in extreme cases, hemorrhage and thrombosis. Renal expression of the transgene was localized to the proximal tubule epithelium, and was associated with tubulointerstitial fibrosis, characterized by excessive collagen deposition and increased fibronectin and plasminogen activator inhibitor-1 immunoreactivity. Pronounced glomerulosclerosis was evident, and hydronephrosis developed with low penetrance. Expression of TGF-beta1 in white and brown adipose tissue resulted in a lipodystrophy-like syndrome. All white fat depots and brown fat pads were severely reduced in size, and exhibited prominent fibroplasia. This reduction in WAT was due to impaired adipose accretion. Introduction of the transgene into the ob/ob background suppressed the obesity characteristic of this mutation; however, transgenic mutant mice developed severe hepato- and splenomegaly. These studies strengthen the link between TGF-beta1 expression and fibrotic disease, and demonstrate the potency of TGF-beta1 in modulating mesenchymal cell differentiation in vivo.
We have investigated the ability of exogenous transforming growth factor-beta (TGF-beta) to induce osteogenesis and chondrogenesis, critical events in both bone formation and fracture healing. Daily injections of TGF-beta 1 or 2 into the subperiosteal region of newborn rat femurs resulted in localized intramembranous bone formation and chondrogenesis. After cessation of the injections, endochondral ossification occurred, resulting in replacement of cartilage with bone. Gene expression of type II collagen and immunolocalization of types I and II collagen were detected within the TGF-beta-induced cartilage and bone. Moreover, injection of TGF-beta 2 stimulated synthesis of TGF-beta 1 in chondrocytes and osteoblasts within the newly induced bone and cartilage, suggesting positive autoregulation of TGF-beta. TGF-beta 2 was more active in vivo than TGF-beta 1, stimulating formation of a mass that was on the average 375% larger at a comparable dose (p less than 0.001). With either TGF-beta isoform, the dose of the growth factor determined which type of tissue formed, so that the ratio of cartilage formation to intramembranous bone formation decreased as the dose was lowered. For TGF-beta 1, reducing the daily dose from 200 to 20 ng decreased the cartilage/intramembranous bone formation ratio from 3.57 to zero (p less than 0.001). With TGF-beta 2, the same dose change decreased the ratio from 3.71 to 0.28 (p less than 0.001). These data demonstrate that mesenchymal precursor cells in the periosteum are stimulated by TGF-beta to proliferate and differentiate, as occurs in embryologic bone formation and early fracture healing.
The effect of transforming growth factor-beta (TGF beta) on bone in vivo was examined. Twelve daily injections of 1 microgram TGF beta directly onto the periostea of parietal bones of neonatal rats stimulated the formation of periosteal woven bone. The thickness of the treated parietal bones increased at least 2-fold in a dose-dependent manner. This TGF beta effect was localized at the sites of injection, and no change was observed in contralateral parietal bones and tibiae. The body weight in these growing rats was not affected by TGF beta 1. TGF beta 2 had effects similar to those of TGF beta 1 on the parietal bones in vivo. These results reveal for the first time that TGF beta stimulates bone formation in vivo and indicate its anabolic role in local bone metabolism.
The ability to transforming growth factor-beta 1 (TGF-beta 1), to stimulate bone healing was evaluated in a rat critical calvarial defect model. Both a low dose and a high dose of TGF-beta 1 were incorporated into two different types of implants: one made from a composite of poly(lactic-co-glycolic acid) (PLPG) (50:50) and demineralized bone matrix (DBM), and the other from calcium sulfate (CaSO 4). Scanning electron microscopy showed that the CaSO 4 implants were more porous than the PLPG/DBM samples. Both types of implants released biologically active TGF-beta 1 for over 300 h in vitro. The samples were implanted in a 9-mm diameter rat calvarial defect for 6 weeks along with contralateral control implants containing no TGF-beta 1. Microradiography and histological analysis were used to assess the bone healing in the defects. Microradiography revealed that the greatest amount of calcified bone (67.5%) was present in in the CaSO 4 implants containing a high dose of TGF-beta 1 while minimal new bone formation occurred in the PLPG/DBM implants. Histologically, the PLPG/DBM implants exhibited an inflammatory response with little mineralization or bone formation. The defects containing the PLPG/DBM implants consisted of a connective tissue stroma with large void spaces. Giant cells and numerous polymorphonuclear leukocytes were present throughout the implants. In contrast, the CaSO 4 implants had only a few inflammatory cells and the presence of mineralization and true bone was a more consistent feature. These preliminary studies show that TGF-beta 1 is capable of inducing new bone formation. Furthermore, the materials used to deliver the growth factor can play a significant role in the bone healing process.
Unloaded cylindrical grit-blasted titanium (Ti-6A-4V) implants (6×10 mm) coated with hydroxyapatite ceramic were inserted into the proximal part of the humerus of 20 skeletally mature Labrador dogs. The implants were initially surrounded by a 2 mm gap. In 10 dogs, HA-coated implants without growth factor were inserted in one humerus and implants with 0.3 ug rh.TGF-61 adsorbed onto the HA coating were inserted in the contralateral humerus. In another group of 10 dogs, a dose of 3.0 mg rhTGF-β1 was tested in a similar design. All dogs were killed at 6 weeks after treatment. Results were evaluated by histomorphometry and mechanical push-out testing. Bone ongrowth was increased by one third, using the 0.3 mg rhTQF-β1 stimulation. Bone volume in the gap and mechanical testing showed no statistically significant differences between control and rhTGF-β1 stimulated implants. RhTGF-β1 only moderately enhanced bone ongrowth to hydroxyapatite-coated implants.
Renal fibrosis in mice treated with exogenous human recombinant transforming growth factor-β2.Background
The biologic responses to transforming growth factor-β (TGF-β) suggest many potential therapeutic applications; however, in the only clinical trial to examine the effect of the systemic administration of a TGF-β isoform, patients experienced significant but reversible declines in renal function. We studied the effects of administering human recombinant TGF-β2 to adult mice.Methods
The effect of daily administration of TGF-β2 on tissue vasoconstriction, tissue levels of endothelin and angiotensin II, tissue hypoxia, and renal fibrosis were examined.ResultsDaily administration of TGF-β2 at 10 or 100 μg/kg caused apparent tissue vasoconstriction that was visualized by vascular casting, with the largest impact seen in the kidney. Tissue levels of endothelin 1 and angiotensin II were significantly elevated in kidneys of treated mice, as was urinary thromboxane β2. Renal fibrosis was observed in the cortical tubular interstitium and vasculature, particularly at the cortical-medullary junction and medullary vasa recta; however, glomerular sclerosis was not observed. Fibrosis was correlated to focal tissue hypoxia as determined by immunohistochemical detection of tissue bound pimondazole.Conclusion
We conclude that there are significant histopathologic consequences, focused in the kidney, resulting from the daily administration of high doses of human recombinant TGF-β2, and we propose that selective vascular constriction with consequent tissue hypoxia is a contributing factor.
Although bone has a remarkable capacity for regenerative growth, there are many clinical situations in which the bony repair process is impaired. TGF-β1 is a 25 kD homodimeric protein which modulates the growth and differentiation of many cell types. The ability of TGF-β1, to promote bone formation suggests that it may have potential as a therapeutic agent in disease of bone loss. However, there still exists a need for an effective method of delivering TGF-β1 to the site of an osseous defect for the promotion of bone healing. This paper describes a novel biodegradable controlled release system for TGF-β1 comprised of poly (DL-lactic-co-glycolic acid) (PLPG) and demineralized bone matrix (DBM). The amount and activity of TGF-βI released was determined using several methods including I-labeled TGF-β1 as a tracer, an enzyme linked immunosorbent assay (ELISA) and a growth inhibitory assay (GIA). Protein was released from the devices for time periods of more than 600 h. The amount of TGF-β1 released was directly proportional to both the TGF-β1 loading and the weight percent of DBM in the device. The release kinetics could be further controlled by applying polymeric coatings of varying porosity to the devices. The GIA indicated that between 80 and 90% of the TGF-β1 released from the delivery system retained its bioactivity. The PLPG and DBM existed in phase separated domains within the device as determined by differential scanning calorimetry. Scanning electron microscopy suggested that the devices were sufficiently porous to allow bone ingrowth.
Critical-sized defects (CSDs) were prepared in 60 rabbit skulls, divided evenly among five treatments and two time periods. The treatments consisted of rabbit demineralized bone matrix (DBM) and four different doses of transforming growth factor-β1 (TGF-β1) (0, 0.4, 4, and 40 μg) delivered in 3% carboxymethyl cellulose and Gelfilm. Quantitative assessments of bone formation in the CSDs were accomplished with computerized radiomorphometry and histomorphometry. Results indicated that rabbit DBM and 40 μg TGF-β1 promoted more new bone formation at 4 weeks than the other treatments. By 8 weeks, bone formation in the CSDs was equivalent for all but the 0-μg dose of TGF-β1. The results from the study suggest a single therapeutic dose of TGF-β1 delivered in an appropriate carrier to a critical-sized intraosseous wound should promote bone formation.
A simple method for preparing undecalcified thin sections of bone with implants has been developed. After exposing a surface of bone and implant in a plastic block by sawing thick sections, the surface is stained prior to making a thin section. A glass coverslip is affixed with a thin layer of cement to the stained surface to stabilize the tissue and implant during sectioning. A mixture of glycerine and water is used as a coolant and lubricant. The orientation in situ is preserved allowing demonstration of bone architecture and cells, and the tissue-implant interface. © 1988 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
Transforming growth factor-beta1 (TGF-beta1) plus demineralized bone matrix (DBM) will reconstruct a critical mandibular defect devoid of periosteum in a canine model.
Randomized, blinded, placebo-controlled, prospective animal pilot study.
Canine critical mandibular defects devoid of periosteum were reconstructed with DBM (group 1, n = 3) and DBM plus TGF-beta1 (250 microg TGF-beta1/g DBM) (group 2, n = 3). Radiologic, histologic, and biomechanical testing was performed on the test group and control group specimens at 12 weeks after implantation.
A palpable bone bridge was present in the group 2 subjects 5 to 6 weeks after implantation and was never present in the group 1 subjects. Radiologic and histologic examination at the time of harvest (12 weeks after implantation) demonstrated a solid bone bridge in the group 2 subjects and a fibrous union in the group 1 subjects. Group 2 specimens demonstrated failure in four-point bending testing at an average maximum moment of 9.9 +/- 2.2 N-m. This value was 9.4% of the maximum moment of the contralateral nonoperated side. Group 1 specimens were palpably flexible on plate removal and had a biomechanical strength of 0. The difference in strength between group 1 and group 2 was statistically significant (P < 0.02), supporting the hypothesis that the addition of TGF-beta1 to the DBM carrier resulted in the formation of significantly stronger bone in the critical gap.
The addition of TGF-beta1 to DBM results in healing of a critical bone defect devoid of periosteum in a higher mammalian model.
The aim of this study was to investigate the synergetic influence of surface topography and chemical composition of oral implant materials on bone response. For the experiment screw designed implants were used. The implants were grit-blasted with TiO2 particles. The implants were left uncoated (Ti) or provided with three different amorphous/crystalline Ca-P magnetron sputter coatings, resp. 0.1 μm (CaP-0.1), 1 μm (CaP-1) and 4 μm (CaP-4), in thickness. The implants were inserted in the medial femoral condyles of 12 goats. Each femur received 2 implants. After implantation periods of 6 and 12 weeks the implants were retrieved and prepared for histological and histomorphometrical evaluation (bone contact and bone mass). The light microscopy revealed that bone response to CaP-4 and CaP-1 implants was similar. For example, after 12 weeks, screw threads were almost completely covered with bone. In contrast to CaP-0.1 and Ti implants, where bone apposition was less pronounced. Histomorphometry demonstrated that the bone-to-implant contact for the CaP-1 and CaP-4 implants was significantly higher (P<0.05) than for the CaP-0.1 and Ti implants. This difference existed already after 6 weeks and was even enhanced after 12 weeks. The bone mass measurements revealed that only at 12 weeks CaP-4 implants had significantly more bone contact inside the screw threads than non-coated Ti-implants (P<0.05). Supported by our findings, we conclude that the additional application of a 1–4 μm thick Ca-P magnetron sputter coating can further improve the healing response to surface roughened oral implants placed into trabecular bone.
Polymer implant discs composed of 50:50 poly dl-lactide-co-glycolide (molecular weight about 9000) were used to repair 5mm calvarial defects in 2kg rabbits and osseous repair compared to spontaneous healing (control). After 4 weeks the implants had undergone substantial degradation with little evidence of residual polymer. The extent to which the defects had been replaced by bone showed individual variation. In some animals a layer of bone with normal cancellous architecture had bridged the defect, but at no time was bone observed in intimate contact with the polymer matrix, suggesting that the material had acted as a tissue spacer rather than an osteoconductive substrate. Non-osseous tissue consisted of a highly vascular fibrous connective tissue containing variable numbers of inflammatory cells. In some sites numerous macrophages and multinucleate giant cells were observed, the majority of which were shown by immunocytochemistry to be MHC class ll-positive. Histomorphometric analysis demonstrated no statistically significant difference in osseous repair between control and polymer implant groups after 1, 2 or 3 months. Incorporation of bone matrix proteins extracted from bovine cortical bone into the discs, however, provoked a cellular and humoral immune response which had a significant inhibitory effect on osseous repair. These data suggest, first, that while synthetic polymers have potential as bone graft substitutes, improvements in their performance in vivo are needed and, second, it is advisable to use allogeneic proteins in rabbit models of bone regeneration.
The purpose of the present study was to create an effective bone-graft substitute for the treatment of a diaphyseal nonunion.
A standardized nonunion was established in the midportion of the radial diaphysis in thirty mongrel dogs by creating a three-millimeter segmental bone defect (at least 2 percent of the total length of the bone). The nonunion was treated with implantation of a carrier comprised of poly(DL-lactic acid) and polyglycolic acid copolymer (50:50 polylactic acid-polyglycolic acid [PLG50]) containing canine purified bone morphogenetic protein (BMP) or recombinant human transforming growth factor-beta (TGF-beta1), or both, or the carrier without BMP or TGF-beta1. Five groups, consisting of six dogs each, were treated with implantation of the carrier alone, implantation of the carrier with fifteen milligrams of BMP, implantation of the carrier with 1.5 milligrams of BMP, implantation of the carrier with fifteen milligrams of BMP and ten nanograms of TGF-beta1, or implantation of the carrier with ten nanograms of TGF-beta1. At twelve weeks after implantation, the radii were examined radiographically and the sites of nonunion were examined histomorphometrically.
We found that implantation of the polylactic acid-polyglycolic acid carrier alone or in combination with ten nanograms of TGF-beta1 failed to induce significant radiographic or histomorphometric evidence of healing at the site of the nonunion. The radii treated with the carrier enriched with either 1.5 or fifteen milligrams of BMP showed significantly increased periosteal and endosteal bone formation on histomorphometric (p < 0.05) and radiographic (p < 0.02) analysis.
Bone formation in a persistent osseous defect that is similar to an ununited diaphyseal fracture is increased when species-specific BMP incorporated into a polylactic acid-polyglycolic acid carrier is implanted at the site of the nonunion. TGF-beta1 at a dose of ten nanograms per implant did not induce a similar degree of bone formation or potentiate the effect of BMP in this model.
The biodegradable implant containing BMP that was used in the present study to treat diaphyseal nonunion is an effective bone-graft substitute.
TGF-beta 1 is a potent inhibitor of hepatocyte proliferation in vivo and in culture and an inducer of fibrogenesis. It is produced by non-parenchymal cells in normal, regenerating, neoplastic and pre-neoplastic liver. TGF-beta 2 and beta 3 are also found in liver non-parenchymal cells and the amounts of their mRNAs increase during liver regeneration. TGF-beta 2 has similar effects to TGF-beta 1. Membranes from normal adult rat liver bind TGF-beta 1 with kinetics consistent with the presence of a single high affinity binding site; membranes from livers that have been regenerating for 12-72 hours show high affinity binding sites not detected in livers of normal or sham-operated rats. Affinity labelling of membranes from normal and regenerating liver shows two receptor proteins with Mr 85,000 and 65,000. In contrast, a prominent band corresponding to a binding protein of Mr 280,000 is detected in membrane preparations of cultured liver epithelial cells. Although modulation of TGF-beta 1 receptors occurs during liver regeneration, it has not been possible to determine which receptor is responsible for the TGF-beta 1 effects in hepatocytes. Other studies have demonstrated a significant correlation between TGF-beta 1 mRNA expression and various indicators of fibrogenesis in patients with chronic liver disease. Thus in animals and humans TGF-beta 1 appears to play a major role in the pathogenesis of fibrosis in chronic liver disease.
Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional regulatory protein. It is capable of inducing site-specific healing responses by increasing collagen synthesis and deposition as well as remodeling at sites of soft tissue repair. Large bony defects in the skull heal by fibrous connective tissue and never form bone unless osteoinductive bony fragments or powders are placed in the defect. We have found, however, that the single application of human recombinant TGF-beta 1 in a simple 3% methylcellulose gel to skull defects induced a dose-dependent increase in intramembranous bone formation. Complete bony bridging of defects occurred within 28 days after treatment with 2 micrograms TGF-beta 1. Sites treated with vehicle alone did not heal with bone formation but rather contained dense fibrous connective tissue between the defect margins.
A simple method for preparing undecalcified thin sections of bone with implants has been developed. After exposing a surface of bone and implant in a plastic block by sawing thick sections, the surface is stained prior to making a thin section. A glass coverslip is affixed with a thin layer of cement to the stained surface to stabilize the tissue and implant during sectioning. A mixture of glycerine and water is used as a coolant and lubricant. The orientation in situ is preserved allowing demonstration of bone architecture and cells, and the tissue-implant interface.
Implantation of demineralized diaphyseal bone matrix in subcutaneous sites induces a sequence of events resulting in the local differentiation of endochondral bone. Demineralized bovine bone matrix was dissociatively extracted in 4.0 M guanidine hydrochloride and the bone-inductive proteins were purified greater than 12,000-fold. The purification steps include affinity chromatography on heparin-Sepharose, hydroxyapatite chromatography, gel filtration, and C18 reverse-phase HPLC. Since the purified protein in conjunction with insoluble collagenous bone matrix induced new bone differentiation in vivo we have designated this component osteogenin. The osteogenic potential is specific for osteogenin and is not exhibited by previously isolated growth factors.
Enhancement of bone ingrowth with transforming growth factor-beta was evaluated in a canine model. Ten dogs had bilateral implantation of a titanium-fiber-metal-coated rod in the proximal part of the humerus. A three-millimeter gap between the outer surface of the porous coating and the surrounding cancellous bone was created to impair bone ingrowth. All of the implants were plasma-flame-sprayed with hydroxyapatite and tricalcium phosphate. In each animal, one implant was also treated with recombinant transforming growth factor-beta 1 while the other implant, which was not so treated, served as a paired control. Two doses of transforming growth factor-beta 1 were used: 335 micrograms in five animals and 120 micrograms in the other five. At four weeks, the amount of bone ingrowth in the implants that had been treated with 120 micrograms of transforming growth factor-beta 1 was threefold higher than that in the paired controls (p = 0.009), but with the numbers available there was no significant increase in bone ingrowth with the higher dose. The amount of new-bone formation in the three-millimeter gaps adjacent to the treated implants was twice that in the gaps of the paired controls, regardless of the dose. The differences between the treated and control implants with regard to the architecture of the new bone in the gap indicate that the mechanism of action of transforming growth factor-beta 1 may include both proliferation of osteoprogenitor cells and production of matrix by committed osteoblasts. Compared with the findings in a previous study in which this canine model was used, the data from the present investigation indicate that enhancement of bone ingrowth in implants that have been treated with a combination of a hydroxyapatite-tricalcium phosphate coating and transforming growth factor-beta 1 may exceed that obtainable with grafting of the gap with autogenous cancellous bone.
The association of a biodegradable material and a growth factor could be of clinical value for treating bone defects. We therefore tested the association of transforming growth factor beta (TGF-beta 1) in fibrin glue and coral granules to heal skull defects in rabbits. Adult rabbits underwent a double trepanation symmetrically in both parietal bones. Using histomorphometry, we compared bone repair after 1 month in control animals (n = 5) and in animals treated with either TGF-beta 1 as a single injection of 1 microgram in methylcellulose (n = 5) or in fibrin glue (n = 5), or with coral granules in fibrin glue (n = 4) or with coral granules and TGF-beta 1 1 microgram in fibrin glue (n = 5). We measured the diameter of the remaining defect and the surface of the bone growth. TGF-beta 1 without coral in either methyl cellulose or fibrin induced a partial closure of the defect as assessed by a significant decrease in the defect diameter, compared with the control group. However, the association of TGF-beta 1 in fibrin and coral induced an area of the bone growth higher than in any other groups (P < 0.05). Two months after surgery, this triple association induced a better healing of the defect than coral alone or control group. In each group treated with TGF-beta 1, the mineralization rate was increased not only at the treated side but also in the contralateral defect which was untreated, suggesting a diffusion of the growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)
Although bone has a remarkable capacity for regenerative growth, there are many clinical situations in which the bony repair process is impaired. TGF-beta 1 is a 25 kD homodimeric protein which modulates the growth and differentiation of many cell types. The ability of TGF-beta 1 to promote bone formation suggests that it may have potential as a therapeutic agent in disease of bone loss. However, there still exists a need for an effective method of delivering TGF-beta 1 to the site of an osseous defect for the promotion of bone healing. This paper describes a novel biodegradable controlled release system for TGF-beta 1 comprised of poly (DL-lactic-co-glycolic acid) (PLPG) and demineralized bone matrix (DBM). The amount and activity of TGF-beta 1 released was determined using several methods including 125I-labeled TGF-beta 1 as a tracer, an enzyme linked immunosorbent assay (ELISA) and a growth inhibitory assay (GIA). Protein was released from the devices for time periods of more than 600 h. The amount of TGF-beta 1 released was directly proportional to both the TGF-beta 1 loading and the weight percent of DBM in the device. The release kinetics could be further controlled by applying polymeric coatings of varying porosity to the devices. The GIA indicated that between 80 and 90% of the TGF-beta 1 released from the delivery system retained its bioactivity. The PLPG and DBM existed in phase separated domains within the device as determined by differential scanning calorimetry. Scanning electron microscopy suggested that the devices were sufficiently porous to allow bone ingrowth.
The temporal dynamics of bone repair in a skull defect in rabbits was examined to characterize the in vivo cellular events occurring following a single local application of recombinant human TGF-beta 1 (rhTGF-beta 1). Rabbits received vehicle or 0.4, 1, 2, or 5 micrograms rhTGF-beta 1 applied to 12 mm defects at the time of surgery. The defect sites were subsequently evaluated by radiography and qualitative and quantitative histology at time points ranging from 1 to 180 days. Based on radiographic assessment, the defect area decreased rapidly in a dose-dependent manner through 35 days after surgery in the rhTGF-beta 1-treated groups. Minimal closure occurred in sites administered vehicle control at all time points examined. Sites treated with rhTGF-beta 1 were characterized histologically by an increase in parameters of active bone formation through 49 days, including percentage osteoid surface, percentage osteoblast/total surface, and an increase in the trabecular bone volume. Bone resorption parameters were increased at 16 and 49 days with histologic evidence of remodeling from woven to lamellar bone. By 70 days, no differences were observed among the groups for parameters of either bone formation or resorption. Bone formation rate was not altered with rhTGF-beta 1 treatment at any time point. These results indicate that exogenously applied rhTGF-beta 1 stimulated the recruitment and proliferation of osteoblasts at the defect site, resulting in a rapid deposition of bony matrix, with normal remodeling processes occurring thereafter. This study supports the hypothesis that TGF-beta 1 is a potent osteoinductive growth factor in vivo and may have potential application as a therapeutic aid to nonhealing bony defects.
Radiofrequent magnetron sputtering was used to produce calcium phosphate coatings on metal and plastic substrates. Scanning electron microscopy showed that the deposited films had a uniform thickness and a dense columnar structure. Energy-dispersive X-ray analysis, X-ray crystal diffraction and atomic absorption spectrometry demonstrated that the sputtered layer was well-crystallized calcium phosphate ceramic with a Ca/P ratio varying between 1.9 and 2.5. The biocompatibility of the coatings was determined by in vitro and in vivo experiments. It was found that the coatings were biocompatible without any sign of adverse tissue reaction. It was concluded that magnetron sputtering is a promising method for forming a biocompatible ceramic coating onto an implant material. Nevertheless, several problems have to be solved before magnetron sputtering can be used on a routine basis for the production of Ca/P coatings.
Growth of bone into cementless prosthetic components is compromised after revision of failed joint prostheses and by osteoporosis, gaps, and micromotion. We studied the effects of recombinant human transforming growth factor-beta 1 adsorbed on ceramic coated implants on the improvement of mechanical fixation and bone growth on the implant. Unloaded cylindrical grit-blasted titanium alloy implants were inserted bilaterally into both the medial and lateral femoral condyles of 10 skeletally mature mongrel dogs. The implants measured 10 mm in length and 6 mm in diameter and were initially surrounded by a 2 mm gap. One implant had an uncoated titanium surface and three implants were coated with tricalcium phosphate and 0, 0.3, or 3.0 micrograms of recombinant human transforming growth factor-beta 1. The dogs were killed at 6 weeks. Mechanical testing showed a 3-fold increase in fixation for the 0.3 microgram dose of recombinant human transforming growth factor-beta 1 and a 2-fold increase for the 3.0 micrograms dose. Histological analysis of bone growth on the implant demonstrated that maximal stimulation occurred with the 0.3 microgram dose, but bone volume in the gap was maximally stimulated by the 3.0 micrograms dose and increased 2-fold over control values. The majority of tricalcium phosphate was resorbed after the 6-week observation period. This study suggests that recombinant human transforming growth factor-beta 1 adsorbed onto implants coated with tricalcium phosphate ceramic can enhance mechanical fixation and bone growth on the implant. The use of transforming growth factor-beta 1 on ceramic coated prosthetic components may help to improve the functional outcome of cementless total joint replacements.
Transforming growth factor beta 1 (TGF-beta 1) is a polyfunctional regulatory cytokine that has been shown to have roles in extracellular matrix interactions, soft tissue healing, and osteogenesis. Twenty-five microL of recombinant human TGF-beta 1 was added to guanidine-extracted demineralized bone matrix carrier and the implants were used to fill a 14-mm osteoperiosteal critical calvarial defect in New Zealand white rabbit model. The defects were allowed to heal over 4 weeks and the degree of new bone formation was assess by radiodensitometry and undecalcified bone histomorphometry techniques. Implants with TGF-beta 1 showed complete bridging of the gap with new bone in all cases, while the controls showed fibrous tissue repair of the gap with little or no new bone formation. These results demonstrate the ability of TGF-beta 1 to induce new bone in a brief time period in an inactive carrier.
The therapeutic utility of a single application of recombinant human transforming growth factor-beta (hTGF-beta) has not been previously tested in large osseous wounds in primates. Sixteen calvarial defects, 25 mm in diameter, were prepared in four adult male baboons (Papio ursinus). In each animal, three defects were treated with increasing doses of hTGF-beta 1 in conjunction with baboon insoluble collagenous bone matrix as carrier (5, 30, and 100 micrograms of hTGF-beta 1/g of matrix). The fourth defect was implanted with collagenous matrix without hTGF-beta 1 as control. Serial undecalcified sections were prepared from the specimens harvested on day 30. Islands of cartilage and endochondral osteogenesis were found in hTGF-beta 1-treated defects, irrespective of the doses used. Histomorphometry of the defect site showed no significant differences between control and hTGF-beta 1-treated specimens with regard to bone and osteoid volumes. However, analysis of the regenerated tissue in proximity to the defect margins only showed that, on average, greater amounts of bone formed in specimens that were treated with 5 and 30 micrograms of hTGF-beta 1 when compared with controls. This suggests a possible effect on osteoblastic cells originating from the periosteal and endosteal spaces of the severed calvaria. Overall, however, this difference has no therapeutic implications for the healing of large cranial wounds in primates. The present findings indicate that a single application of hTGF-beta 1, in conjunction with collagenous matrix, results in limited chondro-osteogenesis in defects of membranous bone of adult baboons.
The bone response to different plasma-spray and magnetron-sputter calcium phosphate (Ca-P)-coated implants was evaluated in a rabbit animal model. Four types of Ca-P coatings have been investigated: a plasma-spray Ca-P coating (HA-PS), a heat-treated plasma-spray Ca-P coating (HA-PS/ht), an amorphous magnetron-sputter coating (Ca-P-a), and a crystalline magnetron-sputter coating (CA-P-c). Seventy-two specially designed cylindrical implants were inserted in the lateral and medial femoral condyles of 18 New Zealand White rabbits. The four differently coated implants were positioned in one animal according to a split-plot design. After implantation periods of 3, 6, and 9 weeks, the bone-implant interface was evaluated histologically. Besides descriptive light microscopical evaluation, quantitative histomorphometrical measurements were done to determine bone contact and the amount of bone surrounding the implant-bone interface. Light microscopical examination revealed that all types of coatings followed the same process of bone healing. Measurements of bone contact at 6 and 9 weeks did not reveal significant differences between the various coatings. For the amount of bone, in a circular region at a certain distance from the implant, the Ca-P-c-coated implants showed a significantly greater amount of bone after 6 weeks of implantation than did the other three Ca-P coatings. At 9 weeks this difference could no longer be measured. On the basis of these findings we concluded that magnetron-sputtered Ca-P coatings show the same process of bone healing as the plasma-sprayed Ca-P coatings when inserted into the trabecular femoral bone of rabbits.
Unloaded cylindrical grit-blasted titanium (Ti-6A-4V) implants (6 x 10 mm) coated with hydroxyapatite ceramic were inserted into the proximal part of the humerus of 20 skeletally mature Labrador dogs. The implants were initially surrounded by a 2 mm gap. In 10 dogs, HA-coated implants without growth factor were inserted in one humerus and implants with 0.3 microgram rhTGF-beta 1 adsorbed onto the HA coating were inserted in the contralateral humerus. In another group of 10 dogs, a dose of 3.0 micrograms rhTGF-beta 1 was tested in a similar design. All dogs were killed at 6 weeks after treatment. Results were evaluated by histomorphometry and mechanical push-out testing. Bone ongrowth was increased by one third, using the 0.3 mg rhTGF-beta 1 stimulation. Bone volume in the gap and mechanical testing showed no statistically significant differences between control and rhTGF-beta 1 stimulated implants. RhTGF-beta 1 only moderately enhanced bone ongrowth to hydroxyapatite-coated implants.
Tricalcium phosphate (TCP) was combined with amylopectin to form a deliverable carrier paste for recombinant human transforming growth factor beta 1 (rhTGF-beta 1) intended for bone repair applications. Approximately 80% of rhTGF-beta 1 was released from the carrier within 24 h following in vitro incubation in serum. Full biological activity was maintained, suggesting the growth factor was stable in this formulation before and after in vitro release. In vivo efficacy also was assessed, in comparison to a sham control group and a placebo-treated group, using a rabbit unilateral segmental defect model (1 cm). Radiographs of defect sites taken at scheduled intervals and the mechanical testing of treated limbs at 56 days demonstrated a higher incidence of radiographic bone union, in concert with a stronger torque strength, in the rhTGF-beta 1-treated group compared to the placebo group. The short duration of the study and the fact that the model used was not a critical defect may account for the lack of superiority of the rhTGF-beta 1-treated group over the healing of the sham control. The in vivo pharmacokinetics of the growth factor evaluated in the same rabbit model suggested that rhTGF-beta 1 persisted intact at the defect site for more than 21 days. Gamma imaging and radioactivity recovery at defects administered to [131I]- and [125I]-labeled rhTGF-beta 1, respectively, estimated the half-life of rhTGF-beta 1 eliminated from the applied site to be 4-6 days. The present report substantiates the potential of rhTGF-beta 1 and its carrier for treatment of bone defects.
Several members of the bone morphogenetic protein (BMP) and transforming growth factor-beta (TGF-beta) families are molecular regulators of cartilage and bone regeneration, although their actual roles and combined interactions in skeletal repair are poorly understood. The presence of several molecular forms suggests multiple functions in vivo as well as synergistic interactions during both embryonic bone development and regeneration of cartilage and bone in postfetal life. Here we show for the first time that recombinant human transforming growth factor-beta1 (TGF-beta1) induces endochondral bone formation in extraskeletal sites of adult baboons. We also show that TGF-beta1 and recombinant human osteogenic protein-1 (OP-1, bone morphogenetic protein-7) synergize in inducing large ossicles in extraskeletal sites of the primate as early as 15 days after implantation. A single application of OP-1, in conjunction with an insoluble collagenous matrix as carrier (5, 25, and 125 microg/100 mg of carrier matrix) induced bone differentiation in the rectus abdominis of the baboon. This level of tissue induction was raised several-fold by the simultaneous addition of comparatively low doses of TGF-beta1 (0.5, 1.5, and 5 microg), which by itself induces bone formation in the rectus abdominis at doses of 5 microg/100 mg of carrier matrix. Combinations of OP-1 and TGF-beta1 yielded a 2- to 3-fold increase in cross-sectional area of the newly generated ossicles, with markedly elevated key parameters of bone formation, and corticalization of the newly formed bone by day 15, culminating in bone marrow generation by day 30. The tissue generated by the combined application of OP-1 and TGF-beta1 showed distinct morphological differences when compared with OP-1-treated specimens, with large zones of endochondral development and extensive bone marrow formation. At the doses tested, synergy was optimal at a ratio of 1:20 by weight of TGF-beta1 and OP-1, respectively. These results provide evidence for a novel function of TGF-beta1 in the primate and the scientific basis for synergistic molecular therapeutics for the rapid regeneration of cartilage and bone.
TGF-beta is a multifunctional cytokine that regulates many aspects of cellular function, including periosteal mesenchymal cell proliferation, differentiation. This experiment is to study its effects on bone defect repair. A rabbit radial bone defect model was used to evaluate the effect of TGF-beta, which was extracted and purified from bovine blood platelets, on the healing of a large segmental osteoperiosteal defect. A 1.5-centimeter segmental defect was created in the mid-upper part of the radial shaft of adult rabbits. The defect was filled with implant containing TGF-beta that consisted of carrier and bovine TGF-beta. Limbs served as controls received carrier alone. The defects were examined radiographically and histologically at 4, 8, 12, 16 and 20 weeks after implantation. The results showed that in TGF-beta implant group, the defect areas at 12 weeks post operation were bridged by uniform new bone and the cut ends of cortex could not be seen; while in control group, the defects remained clear. Only a small amount of new bone formed as a cap on the cut bone ends. In the experimental group, new lamellar and woven bone formed in continuity with the cut ends of the cortex. An early medullar canal appears to be forming and contained normal-appearancing marrow elements; while the control group displayed entirely fibrous tissue within the defect site. Remnants of the cancellous bone carrier were observed in the control specimen. These data demonstrate that exogenous TGF-beta initiate osteogenesis and stimulate the bone defects repair in animal model.
Bone morphogenetic protein (BMP) is known to require a suitable carrier to induce ectopic bone formation in vivo. Hydroxyapatite ceramics have been reported to be effective in some forms but ineffective in others as a carrier of BMP-induced bone formation. In this study we compare three geometrically different forms of hydroxyapatite to examine their functions as carriers of BMP-induced bone formation. A fraction containing all the active BMPs (BMP cocktail) was partially purified from a 4M guanidine extract from bovine bone by a three-step chromatographic procedure. The BMP cocktail was combined with each of three forms of hydroxyapatite--solid particles (SPHAP), porous particles (PPHAP), and coral-replicated porous tablets (coral-HAP)--and implanted subcutaneously into rats. Both the PPHAP and coral-HAP systems induced osteogenesis 2 weeks after implantation, as evidenced by morphological and biochemical observations. Details of the osteogenetic process were followed by double-fluorescence labeling in the coral-HAP system to confirm bone formation on the surface of hydroxyapatite. However, there was no evidence of osteogenesis or chondrogenesis in the SPHAP system. The results indicate that the geometry of the interconnected porous structure in PPHAP and coral-HAP create spaces for vasculature that lead to osteogenesis while the smooth structure and close contact of particles in SPHAP inhibit vascular formation and proliferation of mesenchymal cells, preventing bone and cartilage formation. It was concluded that the geometrical structure in hydroxyapatite ceramics that induces vasculature is crucial as a carrier for BMP-induced bone formation.
The efficacy of local delivery of recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) to promote bone regeneration, with or without cellular contribution from the periosteum, was evaluated in transosseous defects. Implantation of rhTGF-beta 1 into 5 mm in diameter "critical size defects" in the rat mandible resulted in a dose-dependent (0.1-20 micrograms/defect) bone bridging at both 12 and 24 days, independent of the type of delivery system [3% methyl cellulose gel, porous CaCO3 particles, or poly(lactide-co-glycolide) beads]. The bridging, however, never exceeded 24% at 12 days or 34% after 24 days. In contrast, when access of cells from the periosteum to the defect was prevented by means of microporous expanded polytetrafluoroethylene barrier membranes (GORE-TEX membrane), rhTGF-beta 1 caused a dose-dependent inhibition of bone regeneration. The bioactivity of the growth factor was confirmed by implantation of 5 or 10 micrograms rhTGF-beta 1 in 12 mm in diameter bicortical defects in rabbit calvaria, which resulted in complete bone healing within 28 days, whereas control defects displayed a bridging of 40%-50%. The findings support the concept, based on in vitro experiments by others, that TGF-beta 1 primarily has a proliferative effect on cells already committed to the osteoblastic lineage, but also imply that TGF-beta 1 may be inhibitory to induction of osteogenic cells in vivo.
Transforming growth factor (TGF)-beta2 is a pleiotropic cytokine associated with remissions in multiple sclerosis (MS) and amelioration of allergic encephalomyelitis. We assessed the safety of TGF-beta2 in an open-label trial of 11 patients with secondary progressive (SP) MS. Five patients had a reversible decline in the glomerular filtration rate. There was no change in expanded disability status scale or MRI lesions during treatment. Systemic TGF-beta2 may be associated with reversible nephrotoxicity, and further investigation of its therapeutic potential in MS should be performed with caution.
A 1.5 cm segmental defect in the radius of rabbits was used to compare healing at sites administered TGF-beta, with or without autologous bone marrow, to autogenous cortical bone graft. The carrier for TGF-beta consisted of tricalcium phosphate (TCP) granules and hetastarch. The efficacy of TGF-beta formulations and bone marrow (BM) was compared to autogenous bone, carrier control, and untreated defect sites. Bone measurements taken at necropsy included the anterior-posterior (AP) diameter and medial to lateral (LAT) diameter of the defect; the AP and LAT diameters of both radii measured 1 cm proximal to the distal epiphysis, and the AP and LAT diameters of the mid-shaft of the femora. The bones from each group were subdivided for either histological evaluation or for mechanical testing. Strength (maximum torque), energy, angle of rotation and stiffness were determined for both the treated and contralateral radii. Results of the radiographic, necropsy, and mechanical data for defects administered 1.0 microgram of TGF-beta1 + BM or autogenous cortical bone were similar and indicated superior healing compared to defects left blank or administered the carrier control with or without bone marrow. Defects administered 1.0 microgram of TGF-beta1 + BM or autogenous cortical bone had high mechanical strength relative to the control groups and were characterized histologically as healed primarily with lamellar bone. The results from the defects left blank or administered carrier control were similar and generally characterized by poor healing or nonunion. This study demonstrated substantial equality of healing between 1.0 microgram of TGF-beta1 + BM and autograft indicating that this formulation could function as a substitute for autologous grafts.
The expression of transforming growth factor-beta (TGF-beta) correlates with the incidence of renal glomerular and interstitial injury, however, nothing is known of the effect of these proteins on renal hemodynamics. This study examines the renal hemodynamic and morphologic effects of recombinant human TGF-beta2 in normal male Sprague Dawley rats. Acute infusion of TGF-beta (1.2 microg/kg per min) induced no hemodynamic changes, except for a modest though significant fall in mean arterial pressure. Administering TGF-beta2 at varying doses (20, 100, and 400 microg/kg) for 9 wk caused modest increases in systolic BP and proteinuria and minimal tubular interstitial fibrosis, however, renal hemodynamic end points were not significantly altered. TGF-beta2 (800 microg/kg) was also administered to volume-depleted rats for 7 consecutive days. In contrast to the findings in volume-replete animals, administration of TGF-beta2 to volume-depleted rats caused a marked reduction in GFR and medullary blood flow. Histologic fibrosis of the medullary vasa recta and cortical interstitium was seen, but glomeruli were unaffected. Thus, acute and short-term chronic TGF-beta2 administration did not induce major renal changes in the volume-replete state, however, TGF-beta2 combined with volume depletion caused medullary hypoperfusion and reduced GFR.
The osteogenic activity of porous titanium fiber mesh and calcium phosphate (Ca-P)-coated titanium fiber mesh loaded with cultured syngeneic osteogenic cells was compared in a syngeneic rat ectopic assay model. In 30 syngeneic rats, (Ca-P)-coated and non-coated porous titanium implants were subcutaneously placed either without or loaded with cultured rat bone marrow (RBM) cells. Fluorochrome bone markers were injected at 2, 4, and 6 weeks. The rats were sacrificed, and the implants were retrieved at 2, 4, and 8 weeks post-operatively. Histological analysis demonstrated that none of the (Ca-P)-coated and non-coated meshes alone supported bone formation at any time period. In RBM-loaded implants, bone formation started at 2 weeks. At 4 weeks, bone formation increased. However, at 8 weeks bone formation was absent in the non-coated titanium implants, while it had remained in the (Ca-P)-coated titanium implants. Also, in (Ca-P)-coated implants more bone was formed than in non-coated samples. In general, osteogenesis was characterized by the occurrence of multiple spheres in the porosity of the mesh. The accumulation sequence of the fluorochrome markers showed that the newly formed bone was deposited in a centrifugal manner starting at the center of a pore. Our results show that the combination of Ti-mesh with RBM cells can indeed generate bone formation. Further, our results confirm that a thin Ca-P coating can have a beneficial effect on the bone-generating properties of a scaffold material.
The influence of Ca-P magnetron sputter coated implants on the mechanical anchorage was evaluated in a goat model. Therefore, uncoated and coated screw designed commercially pure titanium TiO2-blasted implants were inserted into the trabecular bone of the femoral condyles of 12 goats. The thicknesses of the coatings were 0.1 micron (CaP-0.1), 1.0 micron (CaP-1) and 4.0 microns (CaP-4). In addition, uncoated TiO2-blasted implants (Ti) were used as control. Evaluation of the interface strength and appearance, using torque test and scanning electron microscopy, was done at implantation periods of 6 and 12 weeks. Although, especially at 6 weeks of implantation, the Ca-P coated implants showed higher failure torque values than the TiO2 blasted implants, the observed differences for type of implant and evaluation period were not significant (P > 0.1). SEM evaluation showed that all implants with failure values of more than 100 N conducted bone growth into their screw threads. In addition, we observed that the fracture plane for the CaP-4 implants was situated at the coating-implant interface or inside the coating. For the CaP-1 and CaP-0.1 the fracture line could not be determined definitely. For the Ti implants, the fracture torque testing resulted in failure at the bone-implant interface. Therefore, we conclude that all implants resulted in a good bonding strength with the surrounding bone. The sputtered Ca-P coatings seemed to improve the initial fixation of the TiO2 blasted implants.
The osteoinductive properties of porous titanium fiber mesh, with or without a calcium phosphate coating and loaded with recombinant human bone morphogenic protein-2 (rhBMP-2) or rhBMP-2 and native bovine BMP (S-300) were investigated in a rat ectopic assay model. A total of 112 calcium phosphate-coated and 112 noncoated porous titanium implants, either loaded with rhBMP-2 and S-300 or loaded with rhBMP-2 alone, were subcutaneously placed in 56 Wistar-King rats. The rats were killed 5, 10, 20, and 40 days postoperatively, and the implants were retrieved. Histologic analysis demonstrated that all growth factor and carrier combinations induced ectopic cartilage and bone formation at 5 and 10 days, respectively. At 20 days, bone formation increased and was characterized by trabecular bone and bone marrow-like tissue. At 40 days, more lamellar bone and hemopoietic bone marrow-like tissue were present. At both times, more bone had been formed in calcium phosphate-coated implants than in noncoated samples. Further, in rhBMP-2 and S-300-loaded specimens, bone formation was higher than in rhBMP-2 only-loaded specimens. In rhBMP-2 only-loaded specimens, bone formation was mainly localized inside the mesh material, whereas in specimens loaded with both rhBMP-2 and S-300, the bone was localized inside and surrounding the titanium mesh. The histological findings were confirmed by calcium content and alkaline phosphatase activity measurements. In addition, all specimens showed osteocalcin expression as early as 5 days postoperatively. Our results show that the combination of titanium mesh with BMPs can induce ectopic bone formation and that this bone formation seems to be similar to "enchondral" ossification. In addition, a thin calcium phosphate coating can have a beneficial effect on the bone-inducing properties of a scaffold material. Finally, rhBMP-2 and native BMP act synergistically in ectopic bone induction.
Bone formation in BMP-loaded CaP-coated titanium fiber mesh
- Vehof
Bone formation in CaP-coated titanium fiber mesh
- Vehof
Development of tricalcium phosphate/amylopectin paste combined with recombinant human transforming growth factor beta 1 as a bone defect filler
- Ongpipattanakul
BMP-induced osteogenesis on the surface of hydroxyapatite with geometrically feasible and nonfeasible structures: topology of osteogenesis
- Kuboki
Transforming growth factor-β1 in calcium phosphate cement stimulates osteotransductivity
- E J Blom
- J Klein-Nulend
- L Yin
- R Wenz
- M A J Van Waas