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Bone Cell Differentiation and Growth Factors
Abstract
Bone morphogenetic protein and bone-derived growth factors are biochemical tools for research on induced cell differentiation and local mechanisms controlling cell proliferation. Bone morphogenetic protein irreversibly induces differentiation of perivascular mesenchymal-type cells into osteoprogenitor cells. Bone-derived growth factors are secreted by and for osteoprogenitor cells and stimulate DNA synthesis. Bone generation and regeneration are attributable to the co-efficiency of bone morphogenetic protein and bone-derived growth factors.
... BMPs are differentiation factors belonging to the transforming growth factor-β (TGF-β) superfamily, and cause mesenchymal cells to differentiate (mature) into bone-and cartilage-forming cells. 15,16 BMPs have been proven to be efficacious in healing of calvarial defects in animal models. 17,18 Although BMPs are composed of various subtypes, only one -rhBMP2 in the commercial form of Infuse® bone graft marketed by Medtronic Sofamor Danek, USA -is approved by the FDA. ...
... In fact, some investigations suggested that the combination of BMP and PRP might be antagonistic. 14,15,30,31 In this experiment, PRP vs control, and PRP + rhBMP2 vs rhBMP2 were compared. The authors found that PRP did not have an added effect on bone regeneration. ...
Objective: This study was designed to investigate the optimal combination of known osteogenic biomaterials with shape conforming struts to achieve calvarial vault reconstruction, using a canine model. Methods: Eighteen adolescent beagles were divided equally into 6 groups. A critical size defect of 6 x 2 cm traversed the sagittal suture. The biomaterials used for calvarial reconstruction were demineralised perforated bone matrix (DBM), recombinant human bone morphogenetic protein-2 (rhBMP2) and autogenous platelet-rich plasma (PRP). The struts used were cobalt chrome (metal) or resorbable plate. The groupings were as follows: 1) DBM + metal, 2) DBM + PRP + metal, 3) DBM + PRP + resorbable plate, 4) DBM + rhBMP2 + metal, 5) DBM + rhBMP2 + PRP + metal, and 6) DBM + rhBMP2 + resorbable plate. Animals were euthanised at 3 months post-surgery. There was no mortality or major complications. Analysis was performed macroscopically, histologically, and with computed tomography (CT). Results: There was complete bony regeneration in the rhBMP2 groups only. Non-rhBMP2 groups had minimal bony ingrowth from the defect edges and on the dural surface, a finding confirmed by CT scan and histology. PRP did not enhance bone regeneration. Shape conformation was good with both metal and resorbable plate. Conclusion: rhBMP2 but not PRP accelerated calvarial regeneration in 3 months. The DBM in the rhBMP2 groups were substituted by new trabecular bone. Shape molding was good with both metal and resorbable plate. Key words: Critical size calvarial defect, Cranial vault reconstruction, Metal struts, Resorbable plates, rhBMP2
... Manufacturing in this manner exposes a type I collagen matrix, which is rich in growth factors such as BMPs (BMP-2, BMP-4, and BMP-7), TGF-β1, TGF-β2, and TGF-β3 (Sawkins et al., 2013, Veillette and McKee, 2007, Urist, 1965, Urist et al., 1983, Reddi, 1998. Evidence suggests that BMP-2 and TGF-β may act on bone marrow-derived stem cells and form new bone in the developing enthesis. ...
... This is most likely due to the limited tendon-bone surface area in a rat model not presenting an environment that is as conducive to healing as large animal models and those that utilise a tendon-bone tunnel (Greis et al., 2001. DBM contains multiple growth factors such as BMPs and TGFs (Sawkins et al., 2013, Veillette and McKee, 2007, Urist, 1965, Urist et al., 1983, Reddi, 1998. It is plausible that these were released slowly with time and were therefore unable to completely exert their effect on the MSCs during the six weeks where enthesis healing was permitted to take place. ...
BACKGROUND: The results of surgery for tears of the rotator cuff are variable, with failure occurring in up to 94% of cases. Demineralised bone matrix (DBM) consists of a collagen scaffold containing multiple growth factors and has been used successfully to improve tendon-bone healing. By combining DBM with stem cells its effects may be enhanced given that many of the growth factors it contains are able to direct stem cell differentiation down tenogenic, chondrogenic, and osteogenic lineages. These cell lines produce elements essential to the formation of a naturally graded enthesis. AIM: To investigate the effect of DBM on regeneration of the tendon-bone interface, and whether its function can be enhanced by mesenchymal stem cells (MSCs). HYPOTHESIS: DBM will improve tendon-bone healing in an enthesis defect model, and its effect may be further enhanced by the incorporation of MSCs. METHODS: The following experiments were undertaken in order to investigate the hypothesis: 1. Tensile testing of allogenic and xenogenic cortical/cancellous DBM. 2. Evaluating the effect of allogenic and xenogenic DBM, incorporated with MSCs, on regeneration of the enthesis in a large animal model of severe tendon retraction. 3. Development of a chronic rotator cuff tear model. 4. Investigation of tendon-bone healing using DBM in a chronic rotator cuff tear model. 5. Investigation of tendon-bone healing using DBM and MSCs in a chronic rotator cuff tear model. RESULTS: Allogenic cortical DBM possessed the greatest tensile strength and was used in vivo to examine tendon-bone healing complicated by retraction. In this, DBM regenerated a direct enthesis characterised by fibrocartilage. A similar effect was noted in a chronic rotator cuff tear model with no additional effect conferred by the stem cells. CONCLUSION: This thesis has shown that DBM can regenerate a fibrocartilaginous enthesis in models of tendon retraction and chronic rotator cuff degeneration.
... It is prepared by pulverization into consistent size followed by decalcification with hydrochloric acid. As a result of decalcification mineral component are lost but retains the Type I collagen, non-collagenous proteins, osteoinductive growth factors including varying concentration of bone morphogenetic proteins (BMPs), growth differentiation factors and other transforming growth factors (TGF-b1, TGF-b2, TGF-b3) (Kale & DiCesare,1995;Urist et al.,1983;Schwartz et al., 2011;Gruskin et al.,2012;Holt & Grainer, 2012). The present study was undertaken to evaluate the haemato-biochemical, radiographic and clinical outcome of femoral fracture healing in dogs treated with open reduction and internal fixation (ORIF) with retrograde intramedullary pinning in conjunction with demineralized bone matrix. ...
... Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org DBM is osteoinductive and osteoconductive biomaterial it might facilitate new bone formation by allowing the cells in the local environment to undergo phenotypic conversion to osteoprogenitor cells and might provide mechanical support for vascular and bone in growth (Urist et al., 1983;Kale & Di Cesare, 1995;Albrek & Johanssen, 2001;Pieske et al., 2009;Schwartz et al., 2011;Gruskin et al., 2012;Holt & Grainer., 2012). The demineralization process of DBM by hydrochloric acid (0.6ml/litre) might enhance the bone morphogenic protein to stimulate local undifferentiated mesenchymal cell to transform into osteoblast (Riley et al., 1996). ...
... An ordered porous structure of the scaffold with large pores of approximately 500 nm size was exhibited, which was suitable for the inward growth of bone cells and tissues [50]. The micro-pores could facilitate cell adhesion, uptake of biological metabolites, and cell proliferation [51,52]. The internal architecture of the scaffold would directly determine the cellular microenvironment and affect cell growth, signaling, and osteogenic differentiation, depending on the cell type [53]. ...
Bacterial infection is a common complication in bone defect surgery, in which infection by clinically resistant bacteria has been a challenge for the medical community. Given this emerging problem, the discovery of novel natural-type inhibitors of drug-resistant bacteria has become imperative. Brucine, present in the traditional Chinese herb Strychnine semen, is reported to exert analgesic and anti-inflammatory effects. Brucine’s clinical application was limited because of its water solubility. We extracted high-purity BS by employing reflux extraction and crystallization, greatly improved its solubility, and evaluated its antimicrobial activity against E. coli and S. aureus. Importantly, we found that BS inhibited the drug-resistant strains significantly better than standard strains and achieved sterilization by disrupting the bacterial cell wall. Considering the safety concerns associated with the narrow therapeutic window of BS, a 3D BS-PLLA/PGA bone scaffold system was constructed with SLS technology and tested for its performance, bacteriostatic behaviors, and biocompatibility. The results have shown that the drug-loaded bone scaffolds had not only long-term, slow-controlled release with good cytocompatibility but also demonstrated significant antimicrobial activity in antimicrobial testing. The above results indicated that BS may be a potential drug candidate for the treatment of antibiotic-resistant bacterial infections and that scaffolds with enhanced antibacterial activity and mechanical properties may have potential applications in bone tissue engineering.
... Urist et al. suggested that there is a hormone-related mechanism that leads to this differentiation. Bone morphologic protein (BMP) has been identified as the possible protein that determines whether a pluripotential mesenchymal cell differentiates to form bone or cartilage instead of muscle or scar [8,9]. ...
Heterotopic ossification (HO) is the process of ectopic bone formation in the periarticular soft tissues and is usually formed in the elbow, hip and knee joint as a complication of trauma, burns, brain injury or surgical procedures. The development of HO around the elbow joint can cause a severe limitation of range of motion (ROM) and may affect daily activities of the patient. Treatment of ectopic bone formation around the elbow is a challenge for many surgeons. Non-operative treatment usually fails to restore the ROM of the elbow joint; thus, surgery is necessary to restore the function of the joint. In the past, many surgeons suggested that a delayed excision of HO, until maturation of the ectopic bone, is the best option in order to avoid any possible recurrence. However, many authors now suggest that this delay may lead to complications such as muscular atrophy and formation of soft tissue contractures that can cause a greater impairment of elbow function; thus, early excision is a better option and can better restore the elbow ROM. We performed a literature research of articles that investigated which is the best time of HO excision and we also evaluated if the tethering effect of HO can lead to a greater impairment of the elbow function. We found numerous studies suggesting that a limitation in ROM of the elbow can appear from the tethering of the ectopic bone formation and not only from primary HO. Concerning the HO excision, there were no significant differences between patients who underwent delayed and early excision, concerning the recurrence rate of HO around the elbow. Patients who underwent early excision had better restoration of elbow ROM; thus, early excision, combined with a rehabilitation program, is reported to be the best option for these patients.
... Bone morphogenetic proteins (BMPs) regulate cellular processes such as differentiation, proliferation, and morphogenesis [1]. They are present within the bone and play a crucial role as inducers of bone formation in cases of bone fractures and bone defects caused by trauma or diseases [2]. ...
Background
rhBMP-2 is regarded as the most potent osteoinductive growth factor, and it has been used in the oral cavity with different carriers. The purpose of this study is to evaluate the bone-regenerative effect of rhBMP-2 delivered with different carrier systems through three-dimensional cone beam computed tomography analysis.
Method
A total of 112 patients underwent oral surgery with rhBMP-2 application (Group 1, n = 53) or without rhBMP-2 application (Group 2, n = 59). Group 1 was divided into 3 groups according to carriers, rhBMP-2 with allograft (Group 1–1, n = 34), rhBMP-2 with xenograft (Group 1–2, n = 5), and rhBMP-2 with absorbable collagen sponge (Group 1–3, n = 14). Cone beam computed tomography scans were taken before surgery (T0) 6 months after surgery (T1). The volume of defects was measured through the three-dimensional image analysis tool.
Results
The average bone regeneration rate of Group 1 was significantly greater than that of Group 2. Within Group 1, the group that used allograft as a carrier (Group 1–1) showed significantly higher bone regeneration rates than the group that used absorbable collagen sponge as a carrier (Group 1–3).
Conclusion
The use of rhBMP-2 after oral surgery results in a superior bone regeneration rate compared to not using rhBMP-2, and its efficacy depends on the carriers it is used with. Allograft affects bone regeneration more than absorbable collagen sponge when it is carried with rhBMP-2. Therefore, the appropriate use of rhBMP-2 with suitable bone grafting materials is useful for promoting postoperative bone regeneration in oral surgery.
... Bone graft materials such as autogenous grafts, demineralized freeze-dried bone allograft (DFDBA), freeze-dried bone allograft (FDBA), bone xenografts, or synthetic bone substitutes have been used in the treatment of intrabony defects over years. [1] Growth factors are important for periodontal regeneration because they are implicated in the proliferation, chemotaxis, and differentiation of various cells such as fibroblasts and osteoblasts. Platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) containing growth factors possibly enhance periodontal regeneration, and the efficacy of these sources has been well established. ...
Background:
The versatile combination of emdogain or enamel matrix derivative (EMD), recombinant human platelet-derived growth factor-BB (rhPDGF-BB), and demineralized freeze-dried bone allograft (DFDBA) has not been utilized in the treatment of intrabony defects yet.
Aim:
The present study attempted to investigate the efficacy of a combination of simple, uncomplicated nature of modified minimally invasive surgical technique (M-MIST) with EMD, rhPDGF-BB, and DFDBA in the surgical management of intrabony defects and to assess the possible favorable effects for a period of 6 months.
Patients and methods:
Thirty healthy subjects were included in the present double-blind, randomized controlled, two-arm parallel study. The test group was treated with M-MIST by using rhPDGF-BB, EMD, and DFDBA, and the control group was treated with M-MIST by using rhPDGF-BB and EMD.
Results:
Differences between the mean values of primary clinical parameters including relative attachment level, probing depth, and gingival recession at baseline and those at 6 months after surgery were statistically significant in both groups. Inter-group comparison for clinical attachment level gain, probing depth reduction, and change in the position of gingival margin revealed no statistically significant differences. Inter-group comparison revealed significant differences in linear bone growth (LBG) and percentage bone fill (% BF) but no significant differences in the residual defect depth and change in the alveolar crest position.
Conclusion:
The additional use of DFDBA provides superior benefits in terms of LBG and % BF in intrabony defects. This improvement might be attributed to the use of an osteoinductive scaffold.
... In this spirit, various multiscale micromechanics approaches, as described in Sec. 3, have been carefully validated by means of biomechanical, biophysical, and biochemical experiments [23,35,47,49,52,55,147]. Typically, such experiments concern (i) "universal", i.e. tissue-invariant, mechanical properties of the elementary components making up any tissue belonging to a particular class of tissues (e.g., in the case of bone tissues: molecular collagen type I (making up around 90% of all organic matter found in bone [148]), hydroxyapatite, and water with non-collagenous organic, see Secs. 4.2-4.4 for details); (ii) tissue-specific mechanical properties at any hierarchical level above that of the elementary constituents, tested across the vertebrate animal kingdom, see Secs. ...
Biological materials and systems are hierarchically organized.The main motivation for hierarchical biomechanics is that the wide variability of mechanical properties encountered at the macroscopic scale may be traced back to just a few universal. i.e. tissue-invariant, mechanical properties of elementary components at a sufficiently small scale (such as collagen, elastin, and water in case of soft tissues; complemented by hydroxyapatite in case of hard tissues), and to the nano and microstructures which the latter build up. This challenging task requires a physically rigorous and mathematically sound basis, as provided by Finite Element and Fast Fourier Transform methods, as well as by continuum micromechanics resting on (semi-)analytical solutions for Eshelby-type matrix-inclusion problems. Corresponding numerical and analytical mathematical models have undergone diligent experimental validation, by means of data stemming from a variety of biophysical, biochemical, and biomechanical testing methods, such as light and electron microscopy, ultrasonic testing and scanning acoustic microscopy, as well as physico-chemical tests associated with dehydration, demineralization, decollagenization, ashing, and weighing in air and fluid. While elastic scale transition and homogenization methods have attained a high maturity level, the hierarchical nature of dissipative (i.e. viscous or strength) properties is still a vibrant field of research. This applies even more to hierarchical approaches elucidating the interface between biological cells and extracellular matrices, and to the highly undiscovered mechanics unfolding within biological cells.
... 66 It also threw light on the known ability of connective tissue cells throughout the body that could be induced into bone formation by osteoinductive factors. [67][68][69][70] These connective tissue cells had been previously designated as 'inducible osteogenic precursor cells' (IOPC) by Friedenstein, 71 as opposed to the term 'determined osteogenic precursor cells' (DOPC), which are cells close to bone surfaces that are committed to osteogenesis without any inductive stimulus being required. ...
... PRP was selected as a source of blood-borne factors because PRP is derived from the blood of each individual and contains high concentrations of various osteogenic cytokines [25][26][27][28]. RhBMP-2 was selected to represent one of the existing bone-derived factors, despite not being from the experimental animals in this study, because bone morphogenetic proteins, BMP-2 in particular, are highly osteoinductive growth factors released from old bone matrix, and a previous immunohistochemical analysis of bone samples surrounding Ti implants revealed high levels of BMP-2 [11,13,[29][30][31][32]. ...
Purpose
Osseointegration consists of bidirectional bone formation around modified implant surfaces by contact osteogenesis and distance osteogenesis. This study tested whether contact osteogenesis on the surface of a modified titanium (Ti) implant is stimulated by cytokines in the blood.
Methods
In the first two types of experiments, sandblasted, large-grit, acid-etched Ti implants and turned Ti tubes were inserted into rabbit tibiae. To exclude the influence of distance osteogenesis, the tubes were inserted into the tibiae, and implants were placed inside the tubes. In a third type of experiment, the implants and tubes were inserted into the rabbit tibiae, and platelet-rich plasma (PRP) or recombinant human bone morphogenetic protein-2 (rhBMP-2) was applied topically. Four weeks after implantation, undecalcified specimens were prepared for histomorphometry. Bone-to-implant contact (BIC) and bone area per tissue (BA) were measured, and the data were analysed using one-way ANOVA at a significance level of 0.05.
Results
When the response of bone to Ti tubes with implants was compared to that without implants (first experiment), little bone formation was found inside the tubes. The mean BIC of implant specimens inside the tubes was 21.41 ± 13.81% in a second experiment that evaluated bone responses to implants with or without Ti tubes. This mean BIC value was significantly lower than that in the implant-only group (without tubes) (47.32 ± 12.09%, P = 0.030). The third experiment showed that rhBMP-2 significantly increased contact osteogenesis on the implant surface, whereas PRP had no effect (mean BIC: 66.53 ± 14.06% vs. 16.34 ± 15.98%, P = 0.004).
Conclusions
Platelet-rich plasma alone is unable to trigger contact osteogenesis on the modified titanium implant surface.
... As polymorphic cells of mesenchymal origin, pericytes partially surround the capillaries [6]. This layer provides progenitor cells for the outer fibrous layer and inner cambium layer and plays an essential role in regulating, supporting, and buffering the reconstruction of bone tissue [9][10][11]. ...
Background
Periosteum plays a significant role in bone formation and regeneration by storing progenitor cells, and also acts as a source of local growth factors and a scaffold for recruiting cells and other growth factors. Recently, tissue-engineered periosteum has been studied extensively and shown to be important for osteogenesis and chondrogenesis. Using biomimetic methods for artificial periosteum synthesis, membranous tissues with similar function and structure to native periosteum are produced that significantly improve the efficacy of bone grafting and scaffold engineering, and can serve as direct replacements for native periosteum. Many problems involving bone defects can be solved by preparation of idealized periosteum from materials with different properties using various techniques.
Methods
This review summarizes the significance of periosteum for osteogenesis and chondrogenesis from the aspects of periosteum tissue structure, osteogenesis performance, clinical application, and development of periosteum tissue engineering. The advantages and disadvantages of different tissue engineering methods are also summarized.
Results
The fast-developing field of periosteum tissue engineering is aimed toward synthesis of bionic periosteum that can ensure or accelerate the repair of bone defects. Artificial periosteum materials can be similar to natural periosteum in both structure and function, and have good therapeutic potential. Induction of periosteum tissue regeneration and bone regeneration by biomimetic periosteum is the ideal process for bone repair.
Conclusions
Periosteum is essential for bone formation and regeneration, and it is indispensable in bone repair. Achieving personalized structure and composition in the construction of tissue engineering periosteum is in accordance with the design concept of both universality and emphasis on individual differences and ensures the combination of commonness and individuality, which are expected to meet the clinical needs of bone repair more effectively.
The translational potential of this article
To better understand the role of periosteum in bone repair, clarify the present research situation of periosteum and tissue engineering periosteum, and determine the development and optimization direction of tissue engineering periosteum in the future. It is hoped that periosteum tissue engineering will play a greater role in meeting the clinical needs of bone repair in the future, and makes it possible to achieve optimization of bone tissue therapy.
... BMP's were first discovered with formation of a completely mineralized woven bone with marrow, ectopically. It was achieved with the experiments done by Marshall Urist [14] using demineralized bone matrix placed in the subcutaneous tissue. ...
Normal healing procedure of bone involves various sequential events to develop bone and bridge the bone -to- bone gap. When this healing occurs with a metal (titanium) fixture on one side, it is called as osseointegration. After extensive studies on this topic, it is found that this procedure occurs in presence of various biologic constituents that are spontaneously released at the site. Thus, to accelerate normal healing after implant placement and make results more predictable, it has been proposed to use these autologous factors in the osteotomy site. Since it is the beginning of a new revolution in dental implantology, right now it is essential to analyze all possible combinations of host conditions, bone quality and quantity and bio factors being used. This can definitely be a boon for the patients with compromised systemic or local conditions.
... In view of these ndings, it has been suggested that contact osteogenesis is initiated by the release of a certain substance from the existing bone 6 . A previous immunohistochemical analysis of bone samples surrounding metal implants revealed high levels of bone morphogenetic protein-2 (BMP-2) 6 , a protein that activates bone healing [13][14][15][16] , suggesting that it may be an initiator of contact osteogenesis. ...
The dental implant relies on osseointegration and the response of bone to the implant surface. This process comprises bidirectional bone formation, including bone deposition on the implant surface toward the existing bone (contact osteogenesis) and vice versa (distance osteogenesis). It is unclear whether these processes are independent or whether contact osteogenesis is initiated by other factors. Therefore, this study aimed to identify the initiator of contact osteogenesis. We hypothesized that contact osteogenesis does not occur when it is physically isolated from distance osteogenesis, which would imply that some factors from the wounded bone normally promote contact osteogenesis. Using a rabbit tibial implant model, we tested the effects of human recombinant bone morphogenetic protein-2 (BMP-2) and plasma-rich plasma, which are possible initiators from bone and blood, respectively. Titanium implants with BMP-2 showed a better bone-to-implant contact (BIC) ratio. We concluded that BMP-2 initiated contact osteogenesis on the surface of titanium implants.
... Cortical bone was chosen as raw material because it produced better clinical results than cancellous bone, and cortical bone contains more bone morphogenetic proteins (BMPs) than cancellous bone per weight unit. BMPs play a crucial role in the initial steps of bone tissue morphogenesis (Urist et al. 1983); however, the ideal size of bone particles used to fill bone defects is ill-defined and a disputed issue Temple and Malinin 2008). A clinical trial is still running to find the ideal bone allograft particle size in grafted extraction sockets and edentulous ridges (Abou-Arraj 2020-2021). ...
During the last two decades autologous platelet and leukocyte rich products (PRP; PRF), opened new perspectives in regenerative medicine. In particular regenerative dentistry played a pioneer role in the application of these products in bone regenerative cases. Many aspects of cytokines, such as, growth factor release, blood cell content and its characterization were reported, but some practical questions are still unanswered in the preparation of PRF membranes and sticky bones. A new folding technique was introduced that created a good quality, pliable, and strong F-PRF membrane with a dense fibrin network and more homogenous blood cell distribution. F-PRF produced a very promising sticky bone combined with human freeze-dried cortical bone matrix gelatin (BMG). There hasn’t been much focus on the quality and character of the applied bone and the optimal membrane/bone particle ratio has not been reported. A 0.125 g BMG/ml plasma (1 g/8 ml) seems like the ideal combination with maximal BMG adhesion capacity of the membrane. Particle distribution of BMG showed that 3/4 of the particles ranged between 300–1000 µ, the remnant 1/4 was smaller than 300 µ. The whole F-PRF membrane and its parts were compared with conventional A-PRF membrane concerning their resistance against proteolytic digestion. The F-PRF was superior to A-PRF, which dissolved within 4–5 days, while F-PRF was destroyed only after 11 days, so this provides a better chance for local bone morphogenesis. The F-PRF pieces had similar resistance to the whole intact one, so they can be ideal for surgical procedures without risk of fast disintegration.
... The common growth factors include insulin-like growth factor, platelet-derived growth factor, and bone morphogenetic protein. Among them, bone morphogenetic protein is firstly isolated and found by Urist as a most widely applied factor (Urist et al., 1983). It is proved that BMP2 and BMP7 are effective in the treatment of bone defects (Sun et al., 2012). ...
The goal of a biomaterial is to support the bone tissue regeneration process at the defect site and eventually degrade in situ and get replaced with the newly generated bone tissue. Biomaterials that enhance bone regeneration have a wealth of potential clinical applications from the treatment of non-union fractures to spinal fusion. The use of bone regenerative biomaterials from bioceramics and polymeric components to support bone cell and tissue growth is a longstanding area of interest. Recently, various forms of bone repair materials such as hydrogel, nanofiber scaffolds, and 3D printing composite scaffolds are emerging. Current challenges include the engineering of biomaterials that can match both the mechanical and biological context of bone tissue matrix and support the vascularization of large tissue constructs. Biomaterials with new levels of biofunctionality that attempt to recreate nanoscale topographical, biofactor, and gene delivery cues from the extracellular environment are emerging as interesting candidate bone regenerative biomaterials. This review has been sculptured around a case-by-case basis of current research that is being undertaken in the field of bone regeneration engineering. We will highlight the current progress in the development of physicochemical properties and applications of bone defect repair materials and their perspectives in bone regeneration.
... The inductive effect of our partially demineralized BP has not been established yet, and the contribution of the degree of demineralization to bone healing is also to be answered. The demineralization of bone matrix has been shown to result in the release of growth factors that induce bone regeneration (Urist, 1965;Urist et al., 1983). However, the BP preparation process includes a heat-treatment step that is thought to result in denaturation of these growth factors (Ohta et al., 2005), thereby limiting their contribution to bone regrowth. ...
Autologous bone grafts (BGs) remain the reference grafting technique in various clinical contexts of bone grafting procedures despite their numerous peri- and post-operative limitations. The use of allogeneic bone is a viable option for overcoming these limitations, as it is reliable and it has been widely utilized in various forms for decades. However, the lack of versatility of conventional allogeneic BGs (e.g., blocks, powders) limits their potential for use with irregular or hard-to-reach bone defects. In this context, a ready- and easy-to-use partially demineralized allogeneic BG in a paste form has been developed, with the aim of facilitating such bone grafting procedures. The regenerative properties of this bone paste (BP) was assessed and compared to that of a syngeneic BG in a pre-clinical model of intramembranous bone healing in critical size defects in rat calvaria. The microcomputed tridimensional quantifications and the histological observations at 7 weeks after the implantation revealed that the in vivo bone regeneration of critical-size defects (CSDs) filled with the BP was similar to syngeneic bone grafts (BGs). Thus, this ready-to-use, injectable, and moldable partially demineralized allogeneic BP, displaying equivalent bone healing capacity than the “gold standard,” may be of particular clinical relevance in the context of oral and maxillofacial bone reconstructions.
... 40 Many growth factors, as BMPs stored in the bone matrix induce the differentiation of mesenchymal cells into osteoprogenitor cells. 41 Demineralized bone particles have generated chondro-induction in vivo from events such as migration of connective tissue cells and their conversion to chondroblasts, production of cartilage extracellular matrix, mineralization of the bone TA B L E 1 Percentage area with respective standard deviation of newly formed bone within the critical defects in the study groups matrix and its invasion by host vessels, resorption of the calcified cartilage matrix and its replacement with bone and hematopoietic marrow. 42 Unlike commercially available processed bone, fresh bone seems to require a much shorter demineralization time, which makes its clinical use feasible. ...
Background:
Bone demineralization has shown to be advantageous in autogenous onlay bone grafts and in pre-osteoblasts cultures, but such procedure has never been evaluated in particulate bone grafts. This study aimed to investigate the role of two demineralizing agents in the repair of the 8-mm critical-size defects in rats' calvaria.
Methods:
Eighty adult male Wistar rats were randomly assigned to one of eight groups as follows: particulate autogenous bone demineralized with citric acid for 15s (CA15), 30s (CA30), or 60s (CA60); particulate autogenous bone demineralized with tetracycline hydrochloride for 15s (TCN15), 30s (TCN30), or 60s (TCN60); blood clot (NC), and non-demineralized autogenous bone (PC). The calvariae were harvested at 30 and 60 postoperative days (n = 5) for blinded histological and histometric analysis of the percentage area of newly formed bone within the defects.
Results:
In the NC and TCN groups, bone formation was limited to the margins of the defects at 30 postoperative days, while complete closure was present in all the specimens from CA15 group. Both at 30 and 60 postoperative days, histomorphometry showed significant higher area of newly formed bone in specimens demineralized with CA than in those demineralized with TCN or non-demineralized (P < 0.05). TCN appeared to impair bone neoformation, as its use produced similar or inferior results compared to blood clot.
Conclusions:
Demineralization of particulate bone grafts with CA during 15s enhanced the regeneration of critical-size defects and may be a promising adjuvant in regenerative procedures. TCN seems to be improper for this purpose. This article is protected by copyright. All rights reserved.
... To develop a bioactive surface, many studies have applied growth factors to improve the osteogenic abilities [33,34] and either antibiotics [15] or inorganic nanoparticles [23] to impart the implant surface with antibacterial properties. Despite the excellent results reported from these studies, there are clearly also challenges [35]. ...
The nanotubular surface of titanium implants is known to have superior osteogenic activity but is also vulnerable to failure because of induced bacterial attachment and consequent secondary infection. Here, the problem was attempted to be solved by depositing nanosized tetracycline (TC)-loaded particles in poly(lactic-co-glycolic acid) on titania nanotubes (TNTs) using the electrospray deposition method. The antibacterial effect of the newly formed TNT surface was considered using the common pathogen Staphylococcus aureus. Maintenance of the biocompatibility and osteogenic characteristics of TNTs has been tested through cytotoxicity tests and osteogenic gene expression/extra-cellular matrix mineralization, respectively. The results showed that TNTs were successfully formed by anodization, and the characterization of TC deposited on the TNTs was controlled by varying the spraying parameters such as particle size and coating time. The TC nanoparticle-coated TNTs showed antibacterial activity against Staphylococcus aureus and biocompatibility with MC3T3-E1 pre-osteoblasts, while the osteogenic activity of the TNT structure was preserved, as demonstrated by osteocalcin and osteopontin gene expression, as well as Alizarin red staining. Hence, this study concluded that the electrosprayed TC coating of TNTs is a simple and effective method for the formation of bactericidal implants that can maintain osteogenic activity.
... Le facteur principal est le facteur de croissance endothélial vasculaire (VEGF) qui contrôle la migration endothéliale, la prolifération et la formation des tubes. Il exerce également des actions sur les cellules osseuses, y compris les ostéoblastes et les ostéoclastes (Urist et al., 1983). D'autres facteurs de croissance exprimés lors du développement des vaisseaux sanguins -comme le facteur de croissance des fibroblastes (FGF), le facteur de croissance transformant (TGFb), les protéines morphogénétiques osseuses (BMP), le facteur de croissance analogue à l'insuline (IGF), le facteur de croissance dérivé des plaquettes (PDGF), etc. (Folkman et Klagsbrun, 1987 ;Bai et al., 2013) -jouent un rôle important dans le processus de formation de remodelage osseux. ...
Aim. Bone healing depends on several systemic and local factors but blood supply is an essential requirement. The mineral supply through the new vascular network after implant placement will depend on the bone quality of the area. This review aimed to describe the importance of vas- cularization for bone formation and how it can influence implant osseointegration.
Material & methods. An electronic and hand search were conducted in Medline-PubMed. All type of studies were included. Only articles related to peri-implant bone forma- tion in the oral cavity were selected. No follow-up limitation was applied.
Results. A total of 10 articles were included according to the inclusion and exclusion criteria. Two groups were created: (a) angiogenesis and (peri-implant) bone formation, (b) vas- cularization of the peri-implant area. Trabecular bone pre- sented a rich vascular network whereas few blood vessels could be seen in the cortical bone, which was also the area that supported the highest stress during masticatory func-tion. Blood vessels density was significantly higher in the group with an interimplant distance of 3 mm versus 2 mm. Moreover, if blood supply was already compromised due to other factors (inter-implant distance, age or edentulous ridge), implant osseointegration was also affected.
... from neighboring tissues. For example, during angiogenesis, a pre-requisite for bone formation, endothelial cells form a basement membrane rich in collagen type IV, among others [25][26][27]. Interestingly, collagen type IV was shown to bind BMP3 [28] and TGFβ1 [29]. ...
Bone Morphogenetic Proteins (BMPs) are extracellular multifunctional signalling cytokines and members of the TGFβ super family. These pleiotropic growth factors crucially promote bone formation, remodeling and healing after injury. Additionally, bone homeostasis is systematically regulated by mechanical inputs from the environment, which are incorporated into the bone cells' biochemical response. These inputs range from compression and tension induced by the movement of neighboring muscle, to fluid shear stress induced by interstitial fluid flow in the canaliculi and in the vascular system. Although BMPs are widely applied in a clinic context to promote fracture healing, it is still elusive how mechanical inputs modulate this signalling pathway, hindering an efficient and side-effect free application of these ligands in bone healing. This review aims to summarize the current understanding in how mechanical cues (tension, compression, shear force and hydrostatic pressure) and substrate stiffness modulate BMP signalling. We highlight the time-dependent effects in modulating immediate early up to long-term effects of mechano-BMP crosstalk during bone formation and remodeling, considering the interplay with other already established mechanosensitive pathways, such as MRTF/SRF and Hippo signalling.
... Marshall Urist identified rhBMP2 in 1965 after experimentation definitively showed its ability to produce ectopic bone within a muscle pocket in a surgical lagomorph model (Canalis, 1985;De Biase and Capanna, 2005;Einhorn, 2003;Ripamonti, 2017;Ronga et al., 2013;Urist, 1965;Urist et al., 1983). Decades of improvement followed with research primarily focused on how to make delivery more efficient in order to augment bone healing and create bone in spaces that previously could only be treated with autologous bone graft, allograft, xenograft, or alloplastic graft. ...
Once thought to have revolutionized therapeutic intervention in surgery, Recombinant Human Bone Morphogenic Protein 2 (rhBMP2) is now in its second decade of sustained controversy over the side effects associated with its use. Side effects associated with clinical use of rhBMP2 (Infuse, Medtronic Inc) include a marked inflammatory response, pain, therapeutic failures, ectopic bone, tissue degradation, and death. What is missing, despite the depth of literature on the subject, is a direct interrogation of rhBMP2, specifically for inflammation. Here we set out to determine if rhBMP2 alters traditional macrophage markers associated with pro-inflammatory responses, and pro-reparative responses to injury. Based on our previous work, we hypothesized there would be no direct effect of the peptide on macrophage polarization. Here we utilized commercially available murine macrophages, RAW 264.7, and treated these cells with rhBMP2 in standard growth media or macrophage polarizing media (M1 and M2) at several doses of the peptide. Our readouts were cell viability, apoptosis, gene expression of M1 and M2 markers, and ELISA for M1 marker iNOS, and M2 marker Arg1. Our data give very little evidence to support an alteration in macrophage phenotype by rhBMP2 alone, or alteration of the phenotype when cultured in enriched M1 or M2 media. These results further suggest that other factors associated with the clinical use of Infuse, likely supraphysiological rhBMP2 doses and off label usage, are more likely the culprit for poor outcomes. This further reinforces the utility of rhBMP2 and other peptides in tissue engineering therapies when conditions are tightly controlled.
... 37,38 Substrate adhesion allows cells to attach and sense the physicomechanical properties of the extracellular matrix (ECM) via transmembrane integrin receptors. [39][40][41] In turn, differentiation of bone progenitors cells is regulated by ECM chemical factors, 42,43 but recent studies suggest that electrical properties (electrical charges) of the ECM might also play an important role on cell signaling and bone repair. [44][45][46][47][48][49] Surface electrical charges and streaming potentials resulting from ionic exchanges can produce electrical cues necessary to regulate cell function. ...
... As 90% of the organic matter in bone is collagen [76], the extracellular volume fraction of collagen follows as (29) denotes the volume fraction of the extrafibrillar space in unmineralized tissue, reading as [66] [1,67] as the minimum diffraction spacing occurring in fully dried unmineralized collageneous tissues. According to the standard geometrical notions of continuum mechanics [77,78], the diffraction spacing gives access to the ratio between the hydrated and the fully dried fibrillar volumes, through ...
Micromechanical representation of bone ultrastructure as a composite of aligned mineralized collagen fibrils embedded in a porous polycrystalline matrix has allowed for successfully predicting the (poro/visco-)elastic and strength properties of bone tissues throughout the entire vertebrate animal kingdom, based on the “universal” mechanical properties of the material's elementary components: molecular collagen, hydroxyapatite, and water-type fluids. We here check whether the explanatory power of this schematic representation might extend beyond the realm of mechanics; namely, toward electrodynamics and X-ray physics. This requires knowledge about the electron density distribution across the bone ultrastructure, reflecting the organization of collagen molecules, hydroxyapatite (mineral) crystals, and water with non-collageneous organics. The latter follow three principal, mathematically formulated, “universal” rules, namely (i) a unique bilinear relationship between mineral and collagen concentrations found in bone tissues throughout the vertebrate animal kingdom, (ii) the precipitation of mineral from a ionic solution under closed thermodynamic conditions, governing mass density-dependent lateral distances between the long collagen molecules, and (iii) the identity of the extracollageneous mineral concentration in the fibrillar and extrafibrillar, as well as in the gap and the overlap compartments of bone ultrastructure. The corresponding electron density distributions are then inserted into Fourier transform-type solutions of the Maxwell equations specified for a Small Angle X-ray Scattering setting. The aforementioned mineral distribution, as well as random fluctuations of fibrils, both within their transverse plane around a hexagonal lattice and in form of axial shifts, turn out to be the key for successfully predicting experimentally observed X-ray diffraction patterns. This marks a new level of quantitative, “mathematized” understanding of the organization of bone ultrastructure. In particular, earlier interpretations of SAXS data, leading to the idea of bone being a soft organic matrix with stiff mineral inclusions, may have been overcome, in favor of a more complex, but also more realistic modeling concept concerning the ultrastructural organization of bone.
... There are three types of bone cell: osteoblasts, osteocytes and osteoclasts; each has a distinct functional role and associated morphology. Osteoblasts are responsible for producing the bone extracellular matrix (ECM), and a variety of factors can induce the formation of pre-osteoblasts (Urist et al., 1983 permeating the entire bone matrix. The surface of these canaliculi represents a significant percentage of total surfaces for exchangeable bone calcium. ...
THE PROCESS OF BONE HEALING is an exquisite and complex phenomenon that requires the interaction of three key ingredients: 1) competent bone-forming cells, 2) a suitable framework or scaffold, and 3) the presence of biological stimulants. The principal actors in the process of bone formation are the bone-forming cells, the osteoblasts, and their precursors, the mesenchymal stem cells (MSC). Bone formation is the direct result of the self-assembly and mineralization of the extracellular matrix (ECM) produced by the osteoblasts. The osteoblast arises from the MSC and progresses through a series of maturation steps, eventually becoming an osteoblast. In this process, the cell stages of the maturation sequence are known by distinct names. The term osteoprogenitor is commonly used to refer to all the cell stages preceding the osteoblast, including the MSC.
The research and production of new nanocomposites and nanolaminates that is suitable for a variety of applications such as surface-modified dental and orthopedic implants and prostheses for enhanced soft and hard tissue attachment and scaffolding materials with increased bioactivity for tissue repair and regeneration has been ongoing. The developments of nanoceramic composite coatings based on calcium phosphate have been the focus for biomedical and dental researchers since 2000. A greater emphasis will be placed on the incorporation of molecular and nanoscale-based biological materials and pharmaceutics such as bone morphogenetic proteins, growth factors, stem cells, osteopontin, and simvastatin into calcium phosphate as multifunctional nanocomposite coatings in an effort to reduce the timeframe needed for implant integration as well as enhancing and promoting osseointegration of dental and orthopedic implants and prostheses.
Recent advances in molecular biology have begun to identify many of the molecular mechanisms involved in the control of cellular differentiation and subsequent growth. However, while this information may ultimately permit manipulation of animal growth it is important to remember that the most essential factor for growth remains adequate nutrition. The high correlation of growth rate with milk intake in neonates reminds us that growth is highly dependent on the rapid establishment of enteral nutrition after birth. The endocrine changes consequent on this, as well as the supply of nutrients to support metabolic homeostasis, play important rôles in determining survival.
Birth is associated with dramatic changes in the secretion and plasma concentrations of many hormones (including insulin, glucagon, growth hormone (GH), triiodothyronine (T 3 ), adrenaline and cortisol among others). However, while changes in secretion at this time may be of great significance for survival and adaptation during the perinatal period, after this it is only changes in the secretion of insulin and GH which appear closely related to neonatal growth. Sixty percent or more of variation in live-weight gain among lambs during the 1st month of life is positively associated with variation in plasma insulin levels, while associations with GH are strongly negative, despite the evidence for essentiality of GH as a promoter of insulin-like growth factor-1 (IGF-1) production. Observations on other species are consistent with those in lambs.
Investigations in foetal lambs have established that insulin's crucial rôle as a primary regulator of anabolic metabolism and growth begins well before birth. However, while birth does not alter this important rôle, the establishment of enteral nutrition changes the way in which its secretion is modulated and increases its rôle as the principal endocrine regulator of glucose homeostasis. Secretion of gastrointestinal hormones in response to the first food plays an important rôle in this, modulating the secretion of insulin and promoting gut development through trophic actions on the mucosa. Insulin itself, also appears to have trophic effects on the liver and is probably an important determinant of nutritionally mediated alterations in hepatic IGF-1 production. Plasma IGF-1 concentrations, like those of insulin, are highly correlated with the rate of postnatal growth, but unlike insulin, the status of plasma IGF-1 as a direct regulator of cellular growth remains controversial. IGF-1 and the large molecular weight proteins to which it is bound in plasma are synthesized by many peripheral tissues as well as the liver and it seems likely that IGF-1, in association with other specific growth factors, acts principally in the extravascular compartment, as a local paracrine/autocrine regulator of cellular differentiation and growth. Whatever the status of the plasma IGF-1 pool its concentration appears to act as a useful index of the rate of tissue growth. While GH is essential for normal postnatal growth and the production of IGF-1, it does not appear rate-limiting in relation to nutritionally determined variations in growth rate. This contrasts strikingly with its rôle in growth manipulation.
Bone morphogenetic proteins (BMPs) are a group of growth factors with the clinical potential to regulate cartilage and bone formation. Functionally active mature recombinant human BMPs (rhBMPs), produced primarily in Chinese hamster ovary (CHO) cells for clinical applications, are considered difficult to express because they undergo maturation processes, signaling pathways, or endocytosis. Although BMPs are a family of proteins with similar mature domain sequence identities, their individual properties are diverse. Thus, understanding the properties of individual rhBMPs is essential to improve rhBMP production in CHO cells. In this review, we discuss various approaches to improve rhBMP production in CHO cells by understanding the overall maturation process, signaling pathways and endocytosis of individual rhBMPs.
Bone substitutes will play a pivotal role in the future of periodontal regeneration. They are synthetically derived or processed from exoskeletons of other species (xenograft) and are an alternative to autogenous or allogeneic bone replacement grafts. Bone substitutes do not provide the cellular elements necessary for osteogenesis, and they cannot be considered osteoinductive, but instead are osteoconductive, providing a scaffold for new bone deposition. Currently, significant decreases in clinical probing depth and attachment levels have been reported with bone substitutes when compared to flap débridement surgery alone for periodontal osseous defects. Reported differences among bone substitutes, autogenous grafts, and allograft materials, occur with respect to histologic outcomes. Overall, probing depth reduction and attachment level gains are similar for all bone replacement grafts.
A very effective method of improving the life quality of patients is having the capacity to repair and engineer new functional tissues and in the last forty years, tissue engineering and surface modifications have the potential to accomplish this goal. Many questions concerning the interactions of nanomaterials with both soft and hard tissues have been addressed by a multi-disciplinary team of scientists, engineers, and surgeons. More importantly, the exciting and potential possibilities associated with the use of nanostructured materials and nanocomposites as body interactive materials that assist the body to heal by promoting the regeneration of tissues, hence restoring physiological functions. During the last decade, nanostructured materials has attracted a considerable amount of awareness in areas such as medicine and dentistry, and the emphasis at the moment is on the manufacture of nanocomposites that are applicable to a variety of biomedical applications such as tissue engineering and regeneration. Nanocomposites can be defined as an assortment of two or more materials where at least one of those materials should be on a nanometer-scale. By utilizing the composite approach and secondary substitution phases, it is possible to produce nanocomposites with mechanical properties similar to those of human cortical and cancellous bone. The in vivo cytotoxicity and biocompatibility have been the primary consideration in determining the long-term success of any biomaterials and biocomposites. Presently, the most widely used nanocomposite for biomedical and dental applications is manufactured using several well-characterized and available bioceramics in conjunction with metals and polymers (both natural and synthetic). This approach is currently being explored in the development of next generation nanocomposites with an expanded range of dental and biomedical applications. The last part of the two-part chapter aims to give an overview into the production and types of bio-nanocomposites being examined for applications in implant dentistry as a surface modification approach to improve bone-implant bonding and as tissue engineering scaffolds in the treatment of bone defects. Lastly, brief insights into the application of marine structures in bone grafts and in the treatment of bone disease are also provided.
In this study, factors that exert in synergy with rhBMP-2 were investigated using diffusion chambers inoculated with bone marrow cells in the peritoneal cavity of rats. rhTGF-betal, rhActivin, ThVEGF and ThPDGF were selected as counterparts of rhBMP-2. rhBMP-2 was employed at 30 pgl chamber, the minimal dosage required for osteogenesis inside of the diffusion chambers. Other factors were incorporated at l0pg/chamber. rhBMP-2 with TGF-beta1 promoted a considerable amount of bone outside ofthe chambers but scarcely induced calcified tissue inside. Furthermore, many aligning TRACPase-positive multinuclear cells were observed along the external surface of the chambers beneath the newly formed bone. The other combinations did not show remarkable synergistic effects except for rhActivin that showed slightly enhanced osteoinduction. The results suggest that coexistence ofTGF-beta and BMP-2 may piay a greater role in osteogenesis from non-specific tissue outside of the chamber than from bone marrow-derived cells inoculated inside of the chamber. Asian J. Oral Maxillofac. Surg. lI : 91-98, 1999
In skeletal surgical procedures, bone regeneration in irregular and hard-to-reach areas may present clinical challenges. In order to overcome the limitations of traditional autologous bone grafts and bone substitutes, an extrudable and easy-to-handle innovative partially demineralized allogenic bone graft in the form of a paste has been developed. In this study, the regenerative potential of this paste was assessed and compared to its clinically used precursor form allogenic bone particles. Compared to the particular bone graft, the bone paste allowed better attachment of human mesenchymal stromal cells and their commitment towards the osteoblastic lineage, and it induced a pro-regenerative phenotype of human monocytes/macrophages. The bone paste also supported bone healing in vivo in a guide bone regeneration model and, more interestingly, exhibited a substantial bone-forming ability when implanted in a critical-size defect model in rat calvaria. Thus, these findings indicate that this novel partially demineralized allogeneic bone paste that combines substantial bone healing properties and rapid and ease-of-use may be a promising alternative to allogeneic bone grafts for bone regeneration in several clinical contexts of oral and maxillofacial bone grafting.
Bone induction is a process readily defined during prenatal development of the human skeleton, a process that represents homodimer and heterodimer BMPs' signaling, a process that requires BMP mediated signal transduction, and a process that continues throughout the lifespan of the individual to regulate skeletal renewal during postnatal growth, maturation, and senescence. Bone induction throughout the life span involves a skeletal homeostasis that likely involves normal age-related changes in the skeleton as well as pathophysiologic changes associated with diseases.¹⁵ BMPs are a family of remarkably similar gene products that in turn closely resemble the TGF-β /inhibin supergene family. Available data indicate that endogenous BMP are multifunctional regulatory proteins that serve as chemotactic agents to recruit bone marrow-derived mesenchymal stem cells to emigrate from the bone marrow to sites of bone formation; enhance cell-cell interactions to form aggregates of chondrogenic and osteogenic precursor cells; synergistically function to enhance the effects of other growth factors, e.g., TGF-β isoforms; and stimulate and enhance ectopic bone formations. On the other hand, BMPs are not the sole inducer involved in initiating bone induction. Rather, BMPs, along with other chemotactic agents, participate in a complicated but precisely regulated process to induce new bone formation.
The recent advent of recombinant DNA technology has enabled protein-engineering strategies to produce rhBMPs in large quantities and thereby overcome one of the major previous constraints for clinical applications of BMP. Clinical studies testing various combinations of BMP homodimers and heterodimers have learned that the heterodimer rhBMP-2/rhBMP-7 is an extremely effective inducer of new bone formation to augment mandibular and/or maxillary bone growth in vivo.
Finally, a number of novel strategies have been developed to deliver BMPs to complex bone fracture or wound sites, such as collagenous, coral (calcium carbonate), and apatite (carbonate and calcium apatites) matrices. Such delivery systems provide effective approaches to obtain optimal dose effects for protracted periods of time and thereby enhance the predictability of new bone formation in defined regions of the craniofacial- oral-dental complex. These advances should additionally nurture the intellectual appreciation of molecular strategies to attain bone growth rather than biomechanical solutions to these orthopedic challenges.
Each animal model has provided insights. Particularly important was the considerable resistance of bone to infection without manip-ulation (no morrhuate, fracture, rod, wax, or prosthesis). Such pertur-bations allow bone infection with much smaller inocula. Typical inocula decreases are 1000 to 10, 000 fold. Staphylococci may have a selective advantage in bone because of specialized or tropic binding, perhaps to cartilage or collagen. Osteoclast-induced resorption of hydroxyapatite might explain the distribution of some osteomyelitis. Increased osteoclast activity could link the susceptible metaphyseal regions, the repetitively traumatized diabetic foot, a history of blunt bone trauma, fracture, and perhaps even nearby soft tissue infection. Diagnosis remains difficult; gaIlium-67 and indium¹¹¹ labeled WBC probably deserve additional investigation. Therapeutic failures in the rabbit and rat models mirror clinical experience. Clindamycin, rifampin, and quinolones are promising. Neither systemic nor local anti-microbial prophylaxis is well studied yet.
Human mesenchymal stem cells (hMSCs), which have the ability to differentiate into osteoblasts, show promise for bone tissue engineering and bone defect treatment. While there are a number of approaches currently available to accomplish this, e.g., utilizing biodegradable materials loaded with the synthetic glucocorticoid osteogenic inducer dexamethasone (DEX), there are still many disadvantages with the current technologies. Here, we generated light-responsive microgels that we showed are capable of loading and releasing DEX in a light-triggered fashion, with the released DEX being able to induce hMSC differentiation into osteoblasts. Specifically, light-responsive poly(N-isopropylacrylamide-co-nitrobenzyl methacrylate) (pNIPAm-co-NBMA) microgels were synthesized via free radical precipitation polymerization and their size, morphology, and chemical composition were characterized. We then went on to show that the microgels could be loaded with DEX (via what we think are hydrophobic interactions) and released upon exposure to UV light. We went on to show that the DEX released from the microgels was still capable of inducing osteogenic differentiation of hMSCs using an alamarBlue assay and normalized alkaline phosphatase (ALP) activity assay. We also investigated how hMSC differentiation was impacted by intermittent DEX released from UV-exposed microgels. Finally, we confirmed that the microgels themselves were not cytotoxic to hMSCs. Taken together, the DEX-loaded light-responsive microgels reported here may find a use for niche clinical applications, e.g., bone tissue repair.
The review covers problems related to development and application of a new type of “smart” medical materials, mainly, for regeneration of bone tissue that are capable of creating additional stimuli influencing the regeneration process. Application of ferroelectric and magnetoelastic materials is discussed, including their use as sensors and actuators. Physical and materials science principles of development, along with examples of using composite magnetoelectric materials of the piezoelectric/magnetoelastic type as magnetically controlled scaffolds creating local electric fields, are analyzed.
The physiology of bone healing, regeneration, and bone transfer is discussed from the dual perspectives of basic physiology and the application to clinical situations of bone transfer. Recommendations are made regarding clinical applications for the site, orientation, and fixation of grated materials. Rationale for the use of vascularized transfers is presented.
Bone defects resulting from congenital anomalies, trauma, metabolic diseases, infections, and tumor removal are formidable clinical challenges for orthopedics and craniofacial surgeries. Bone tissue engineering is aimed to repair bone defects that fail to spontaneously heal. In the conventional tissue-engineering paradigm, combinations of cells and bioactive molecules are seeded onto three-dimensional (3D) biomaterial scaffolds to create an implantable “osteogenic” implant. To date and despite numerous exciting advances in investigative and preclinical models, bone tissue engineering has encountered a multitude of challenges including autologous donor cell deficiencies, immunorejections of delivered cells, excessive costs, and regulatory approval barriers. These hurdles have impeded the translation of innovative bone tissue engineering strategies from the bench to the bedside. Whereas the bone tissue engineering field continues to explore frontiers in investigative and innovative approaches toward bone defect repair, several translational advances have shown great promise toward development into potential clinical therapeutics. First, there are clinical case reports of ingenious reconstructions of challenging appendicular, axial, and craniofacial defects that, despite their origin from conventional tissue-engineering concepts of delivering cells and/or growth factors, may circumvent otherwise stringent regulatory process of cell transplantation therapies, but rather as the point-of-care treatments. However, caution must be exercised to distinguish between cell therapies that have been proven to be efficacious and safe following randomized clinical trials and those that are offered without clinical trials. Second, new or known molecules that demonstrate efficacy in repairing nonhealing bone defects are being developed with the general concept to augment endogenous bone regeneration. Relative to several other musculoskeletal tissues such as cartilage, intervertebral discs, and ligaments and tendons, bone has relatively robust endogenous capacity for repair. Delivery of new or existing osteogenic molecules that augment bone’s intrinsic capacity for self-repair has been termed cell homing, by recruiting endogenous stem/progenitor cells, that may serve as an alternative or adjunctive to cell transplantation. Third, innovative biomaterials and 3D printing offer unprecedented opportunities for bone tissue engineering and are being developed with translational goals to evolve into clinically viable therapeutics. Finally, we highlight clinical successes and opportunities for regenerating appendicular and craniofacial bone defects. In summary, bone tissue engineering continues to offer the only hope for repairing some of the bone defects that fail to heal.
A vitamina D vem sendo uma das principais e mais estudadas substâncias do nosso organismo. Vários estudos apontam para a sua importância não somente no metabolismo ósseo, mas para sua correlação com os demais órgãos e tecidos e suas implicações em doenças não-ósseas. Esse é um fato relevante, visto que historicamente a indicação clássica do uso da vitamina D é para a prevenção de doenças ósseas como osteoporose, osteopenia, entre outras. O confronto de pesquisas e estudos sobre a influência da vitamina D na odontologia vem a cada dia tomando mais força no mercado nacional e internacional, principalmente sobre a relação de osseointegração e qualidade óssea, associada a hipovitaminose ou a hipervitaminose. Tais estudos levam a crer que a vitamina D é uma das mais importantes vitaminas para nosso corpo e participa ativamente no processo de reparo ósseo.
During neurulation, craniofacial structures derived from the first branchial arch are determined to become maxillary, mandibular, and tongue formations. At least four interdependent developmental processes become integrated: (1) allocation of cells into specific lineages (perhaps during gastrulation); (2) regulation of time-dependent differential regulatory and structural gene expressions; (3) positional information resulting in pattern formations; and (4) morphogenesis, histogenesis, and cytodlfferentiation. This review highlights progress toward understanding when, where, and how the one-dimensional genetic information encoded within DNA (deoxyribonucleic acid) is sequentially expressed into the embryonic craniofacial complex. Specifically, how might intrinsic autocrine and/or paracrine regulatory factors control the developmental program for early first branchial arch morphogenesis, histogenesis, and cytodifferentiation. Rules learned from normal development should be useful toward advancing the diagnosis, treatment, prognosis, and prevention of congenital craniofacial malformations.
Nothing is known on the impact of developmental divergence on periodontal tissue regeneration in vertebrate animals. Molecularly, the induction of tooth morphogenesis is highly conserved deploying across animal phyla a constant and reproducible set of gene pathways, which result in morphogenesis of multiple odontode forms and shapes. Genetic mutations positively affect animal speciation via evolving biting and masticatory forces as well as dietary habits selectively imprinted in animal phyla during evolutionary speciation. The geometry of the attachment apparatus of a tooth is important for the interpretation of the induction of cementogenesis with de novo Sharpey's fibres as in thecodonty, ie, a tripartite attachment of alveolar bone, periodontal ligament and cementum. This review addresses the tooth implantation in different animal clades from the fibrous attachment of the Elasmobranch Carcharinus obscurus dusky shark, reviewing the evolution and functional significance of cementum with functionally inserted Sharpey's fibres. In sharks there is a continuous tooth replacement mechanistically supported by the continuously erupting dental lamina. We show that the arching of the continuously erupting dental lamina, a critical step for the selachians’ tooth differentiation, is prominently characterized by transforming growth factor‐β3 (TGF‐β3) expression not only within the dental lamina but also in cellular condensations in the mesenchymal tissues of the erupting tooth. Such findings indicate the pleiotropic multifaceted activity of a highly conserved mammalian gene across genera, masterminding tooth morphogenesis in both selachians and mammals as well as periodontal tissue induction in the non‐human primate Papio ursinus. In P. ursinus, the induction of cementogenesis entails the expression of TGF‐β3 and osteocalcin with fine‐tuning and regulation of bone morphogenetic proteins BMP‐2 and BMP‐7, and upregulation of TGF‐β3. TGF‐β3 autoinduction and upregulation during the induction of cementogenesis and osteogenesis in P. ursinus provide novel insights into the induction of cementogenesis. It is hypothesized that the evolutionary expression and upregulation of the TGF‐β3 gene may provide the mechanistic insights into the induction of extensive cementogenesis as seen in stem mammals and the induction of trabecular‐like cementum formation in mosasaurs’ tooth attachment. Aspidin, the precursor of cementum, was reported to appear 310‐330 million years ago (Ma) in Odontostraci armoured fish. Studies showed that the differentiation of cementum with inserted Sharpey's fibres is also present in lower amniotes such as Diatectomorpha or Diadectidae, the first herbivorous tetrapods, 323 Ma. In mosasaurs, 168‐165 Ma, there is the induction of extensive trabeculation of cementum though nothing is known on the phylogenetic temporo‐spatial evolution of cementum before Diadectidae and stem mammals. The large trabeculations of cementum as seen in the attachment of extinct mosasaurs invocates a pleiotropic capacity of cemental growth previously unknown. The appearance of cementum facing a vascularized and innervated periodontal ligament space with Sharpey's fibres inserting on to mineralized cementum provides a multiform pleiotropic masticatory apparatus adapted to multiple biting and lacerating forces as well as finely tuned and controlled forces beyond mastication and deglutition. The remarkable cementogenesis as seen in stem mammals but particularly in mosasaurs with cemental trabeculations across the ligament space invocates the developmental capacity of cementum. The large cemental trabeculations as seen in mosasaurs and the cemental growth in stem mammals, together with regenerating scenarios in P. ursinus with large seams of cellular cementum and cementoid populated by contiguous cementoblasts indicate the continuous molecular cross‐talk between cementum, newly formed cementoid matrix, cementoblasts and extracellular matrix soluble molecular signals. This molecular cross‐talk may control the biomolecular homeostasis of both cementum and periodontal ligament, including angiogenesis. A further molecular scenario is invocated by the tight and exquisite anatomical relationships between the cementoid surfaces and the newly formed capillaries. The primitiveness of the craniate masticatory mineralized craniofacial apparatus has been controlled by several yet ancestral common genes not lastly the TGF‐β3 gene. The TGF‐β3 might have been responsible for the induction of cementogenesis not only in extant P. ursinus but also in Diatectomorpha and mosasaurs, thus providing continuous evolutionary mechanisms for the induction of tissue morphogenesis across animal phyla for almost a billion years of evolution, epitomizing Nature's parsimony in controlling tissue induction and morphogenesis. TGF‐β receptor II regulates osterix expression via Smad‐dependent pathways indicating that TGF‐β signalling acts as an upstream regulator of osterix during cementoblast differentiation. The presence of morphogenetic signals within the cemental matrix capable of inducing bone formation needs now to be assigned: bone induction initiated by extracted and partially purified cemental matrices may be the result of a slow release of embryonic remnants of osteogenic signals required and deployed during cementogenesis. The cementum may thus rule the periodontal ligament space homeostasis, remodelling and repair by releasing sequestered morphogenetic signals that were deployed during embryogenesis.
Melatonin (MLT), a hormone secreted from the pineal gland, is recognized as a potential candidate for stimulation of bone regeneration. However, because of its hydrophobicity, the administration of MLT to stimulate bone regeneration is difficult. In this study, an inclusion complex of MLT with 2-hydroxypropyl β-cyclodextrin (HP-β-CD) was prepared to improve the water solubility, and the osteogenic differentiation ability of the inclusion complex was investigated in MC3T3-E1 cells. The formation of HP-β-CD/MLT inclusion complex was confirmed by 1H and 13C nuclear magnetic resonance spectroscopy and wide-angle X-ray diffraction. The water solubility of MLT increased linearly upon addition of HP-β-CD because of the formation of the inclusion complex. Additionally, treatment of the cells with HP-β-CD/MLT inclusion complex showed higher uptake amount of MLT than that treated with free MLT. In addition, treatment of MC3T3-E1 cells with HP-β-CD/MLT inclusion complex increased alkaline phosphatase activity and mineralized matrix deposition, compared to that in free MLT-treated and untreated cells. Furthermore, cells treated with HP-β-CD/MLT inclusion complex exhibited higher expression levels of osteogenic differentiation genes than those in the untreated and free MLT-treated cells. Accordingly, these results suggested that inclusion complexation of MLT with HP-β-CD would be a potential formulation for bone regeneration because of its improved solubility and enhanced osteogenic differentiation efficiency.
FL or WISH cells, originating from the human amnion, were grafted intramuscularly into various strains of mice.1.
When grafted, cells of both lines evoked cartilage formation in their vicinity, provided that the animals were pretreated with cortisone (5 mg) or ACTH (36 u).
2.
No host strain differences were found in respect to the cartilage induction.
3.
Cartilage was replaced by bone, tissue, which was gradually resorbed within two months.
4.
Grafting the FL cells into animals treated with hydrocortisone or imuran and into newborn or X-irradiated mice failed to induce cartilage or bone tissue.
A γ carboxyglutamic acid containing protein has been purified from the calcified tissues of several vertebrates. The presence of three γ carboxyglutamic acid residues in the bovine protein was established by alkaline hydrolysis and amino acid analysis, a method based upon studies with synthetic γ carboxyglutamic acid. The identity of γ carboxyglutamic acid in the bovine protein was established by mass spectroscopy on the unknown amino acid isolated from alkaline hydrolysates. The protein is extracted from finely ground bone during demineralization with EDTA, and purified from EDTA extracts by gel filtration over Sephadex G 100 and chromatography on DEAE Sephadex. The protein has a 6800 molecular weight and an isoelectric point of pH 3.7. The amino terminal 15 residues have been determined, and establish that this protein is not a fragment of the γ carboxyglutamic acid containing blood clotting factors. Similar γ carboxyglutamic acid containing proteins also have been purified from bovine dentine, swordfish vertebrae, and human tibia. No γ carboxyglutamic acid can be detected in the calcified cartilage of elasmobranches, in calf epiphyseal growth cartilage, or in tooth enamel. The bovine protein binds strongly to hydroxyapatite but not to amorphous calcium phosphate, and it is a potent inhibitor of hydroxyapatite crystallization from supersaturated solutions of calcium and phosphate.
A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This tissue culture system starting from isolated bone cells is therefore suitable for studies on the structure and function of bone.
Cells dispersed from the chondrocranial portions of fetal rat calvaria proliferated and performed specialized functions during primary culture in a chemically defined medium. Mature cultures were typified by multilayered clusters of redifferentiating cartilage cells. Flattened cells that lacked distinguishing features occupied areas between the clusters. Alkaline phosphate-enriched, ultrastructurally typical chondrocytes within the clusters were encased in a dense extracellular matrix that stained prominently for chondroitin sulfate proteoglycans. This matrix contained fibrils measuring 19 nm in diameter, which were associated with proteoglycan granules that preferentially bound ruthenium red. A progressive increase in the number of cells indicated the proliferation of certain elements in the primary culture. The cells in primary culture were biochemically as well as morphologically heterogeneous since they were found to synthesize type I and type II collagens. Homogeneous populations of redifferentiated chondrocytes were recovered as floating cells and were shown to express the chondrocyte phenotype in secondary culture. Subcultured cells synthesized type II collagen and its precursors almost exclusively and incorporated 35SO4 into proteoglycan monomer and aggregates to a greater degree than the cells in primary culture. The pattern of proteoglycan monomer and aggregate labeling resembled that of intact cartilage segments and bovine articular chondrocytes. Skin fibroblasts harvested from the same rat fetuses failed to proliferate when maintained under identical conditions. Hence, exogenous hormones, growth factors, and protein are not required for chondrocyte growth and maturation.
• Bone generation and regeneration are associated with a bone morphogen that recruits mesenchymal cells for differentiation into bone. Experiments with particulate bone matrix gelatin implanted in multiple-walled diffusion chambers suggest that bone morphogen is a rapidly diffusible molecule, and consists of a noncollagenous bone morphogenetic protein (BMP). When particulate bone matrix gelatin is implanted inside of diffusion chambers constructed of two to five membranes, ranging from 300 to 750 cu μm in total thickness, large deposits of bone develop on the outside. The volumes of the deposits of new bone are inversely proportional to the thickness (or distance) of transmission of the BMP. Transmission for long distances through interstitial fluid can be accounted for by a low molecular mass hydrophobic BMP, disseminated according to the laws of diffusion.(Arch Surg 112:612-619, 1977)
Plasmaα
2HS-glycoprotein is specifically accumulated in calcified tissues. In the present studies this glycoprotein was isolated from plasma and after iodination with iodine-125 was injected intravenously into young rabbits. The tissue distribution and plasma disappearance rate of this radioactively labeled material were determined. Of the various tissues studied, bone showed the greatest retention of labeled glycoprotein expressed as percentage of the injected dose per gram tissue relative to the plasma content.
The rate of loss of iodinatedα
2HS-glycoprotein from plasma was similar to that ofα
2HS-glycoprotein labeled endogenously by using14C-glucosamine or3H-glucosamine. The uptake of exogenously labeled3I-α
2HS-glycoprotein into bone tissue expressed as a percentage of the injected dose was similar to that of endogenously labeled14C-α
2HS-glycoprotein. These results suggest that the125I-labeled material can be used to study further the metabolism ofα
2HS-glycoprotein by bone tissue.
An in vitro organ culture system of embryonic chick tibiae was used to examine bone formation and bone resorption under conditions of stimulation with 10−12 M parathyroid extract. The bones were cultured in a serum-free medium to alleviate any potential interference by serum growth factors. Evidence is presented showing that this bone culture system responds to parathyroid extract with an increase in both bone resorption and bone formation. Although initially there was a decrease in formation, this was followed after 12–16 h by an increase in formation which was correlated with resorption (r = .96, p < .05). These results suggest that the increase in formation was some consequence of the increase in resorption, and as such are consistent with the interpretation that coupling can occur in vitro.RésuméUn système de culture d'organes in vitro de tibias embryonnaires de poulets a été utilisé pour étudier la formation et la résorption osseuses dons des conditions de stimulation par de l'extrait parathyroidien a 10−12M. Les os ont été cultivés dans un milieu sans sérum pour supprimer toute possible interférence avec des facteurs de croissance sériques. Il est démon-tré que ce système de culture osseux répond à l'extrait parathyroïdien par une augmentation à la fois de la résorption et de la formation osseuses. Toutefois, après diminution initiale de la formation, il y a une augmentation après 12 à 16 heures qui est corrélée à l'augmentation de la résorption (r = 0.96, p < 0.05). Ces résultats suggèrent qua l'augmentation de la formation est conséquence de l'augmentation de la résorption, ce qui est compatible avec l'intérpretation selon laquelle le coupling peut intervenir in vitro.
Transitional epithelium of the guinea pig and rabbit and an established WISH cell line, when grafted under the kidney capsule of cortisone treated mice, survived and proliferated, but never induced bone formation. On the contrary, when grafted intramuscularly into the same recipients, readily induced cartilage and/or bone.
Cell lines capable of inducing cartilage/bone (e.g. HeLa, WISH, CLV-J3, CLV-X, CLV-4 and CLV-14) agglutinate in the presence of Concanavalin A because their membranes contain the appropriate receptors. This indicates that these cell lines may possess surface terminal branched carbohydrates with terminal non-reducing -D-glucopyranosyl, -D-mannopyranosyl or -D-fructofuranosyl residues. Cells devoid of osteogenic properties (T-24, L-929, primary cultures of human and mouse fibroblasts) did not agglutinate in the presence of Concanavalin A, probably because either the receptors were masked or absent. Phytohemagglutinin differed from Concanavalin A in so much as it agglutinated both osteogenic and non-osteogenic cell lines.
Implants of bone matrix, capable of inducing new bone formation, were prepared by decalcifying cortical bone in 0.6 N HCl at 2°C for 48 hours. The inductive property could be inactivated by decalcification in 0.6N HCl containing 66.5% ethanol. When implanted into muscle, both the active and inactive implants were invaded by mesenchymal cells. There was no significant difference in the DNA content of active and inactive implants, but only the active implants induced the synthesis of alkaline phosphatase by a new cell population. Alkaline phosphatase activity was present at 5 days, reached a peak at 20 days after implantation, then declined to 15% of the maximum value by the 40th day after implantation. The deposition of calcium in the implants, coinciding with the differentiation of new bone, began about 12 days after implantation, and gradually increased throughout the 40-day experimental period. Neither recalcification of old matrix nor differentiation of bone occurred in the acid-alcohol-inactivated implants. Induction of alkaline phosphatase synthesis occurred in the pre-osseous phase of morphogenesis 5 days before the appearance of calcium deposits and was not directly correlated with the mechanism of calcification.
Cartilage and bone induction evoked by xenogenic grafts of WISH cells possesses neither generic nor species specificity. No induction was obtained in mice when WISH cells were grafted subcutaneously. Human fibroblasts transformed into CLV-J3 epithelioid cells acquire inductive properties. Hydrocortisone used as an immunosupressive in large doses enables WISH cells grafted into mice to survive, but in contrast to cortisone treated animals, small amounts of hyaline cartilage only were obtained and only in half the animals.Die Bildung von Knochen- und Knorpelgewebe, die durch xenogene intramuskulre Transplantate von WISH-Zellen veranlat wird, wurde bei drei verschiedenen Tierarten (Maus, Ratte und Hamster) beobachtet. Subcutane Implantierung von WISH-Zellen in Muse fhrt zu keiner Knorpel- oder Knochenbildung. Menschliche Fibroblasten, die durch Vakzine-Virus zu epithelialen Zellen (CLV-J3) umgewandelt wurden, knnen nach Transplantation zum Entstehen von Knorpel- und Knochengewebe fhren.Grere Dosen von Hydrocortison, als immundepressives Mittel angewandt, ermglicht den in die Muse implantierten WISH-Zellen ein berleben. Die Bildung von jungem Knorpel wurde jedoch nur in 50% der Flle erreicht, im Gegensatz zu Cortison, das in allen Fllen die Knorpel- und Knochenbildung verursachte.L'induction de cartilage et d'os provoque par la greffe xenognique de cellules WISH ne possde aucune spcificit gnrique ou di'espce. Chez la souris, la greffe sous cutane de cellules WISH ne provoque aucune induction. Les fibroblastes humains transforms en cellules pithlioides (CLV-J3) par le virus de la vaccine ont acquis des praprits inductives.L'hydrocortisone forte dose permet, en tant qu agent immunodepresseur, la survie des cellules WISH greffes chez la souris, mais, contrairement ce que l'on observe chezl'animal trait par la cortisone, du cartilage hyalin est obtenu dans la moiti des cas seulement et en petites quantits.
This report describes some general features of chick embryo chondrocyte cultures and some methods for measuring matrix production. It is reported that as cultures grow, the average chondrocyte acquires an increased ability to synthesize matrix components. In part this increased ability is caused by conditioning of the culture medium, since conditioned medium from chondrocyte cultures can rapidly stimulate mucopolysaccharide and collagen synthesis, but not growth. The cells condition the medium by releasing a factor that has the following characteristics: non-dialyzable, heat and trypsin-sensitive, sensitive to treatment with mercaptoethanol, p-chloromercuribenzoate, and periodate, and a molecular weight greater than 30,000 but less than 150,000. The factor is a specialized product of chondrocytes, since it is not made by unexpressed chondrocytes nor by differentiated pigmented retina cells. Conditioned medium acts rapidly (2 hr) to produce a significant stimulation of the incorporation of sulfate into hyaluronidase-sensitive material. This action is not sensitive to treatment with actinomycin D, and it is suggested that conditioned medium might act on the cell surface. The action of conditioned medium factor represents an example of positive feedback of one specialized product made by a particular cell type on the synthesis of other specialized products made by the same cell type.
Three new mesenchymal phenotypes were expressed in cultures of Swiss 3T3 and C3H/10T1/2CL8 mouse cells treated with 5-azacytidine or 5-aza-2'-deoxycytidine. These phenotypes were characterized as contractile striated muscle cells, biochemically differentiated adipocytes and chondrocytes capable of the biosynthesis of cartilage-specific proteins. The number of muscle and fat cells which appeared in treated cultures was dependent upon the concentration of 5-azacytidine used, but the chondrocyte phenotype was not expressed frequently enough for quantitation. The differentiated cell types were only observed several days or weeks after treatment with the analog, implying that cell division was obligatory for the expression of the new phenotypes. Oncogenically transformed C3H/10T1/2CL8 cells also developed muscle cells after exposure to 5-azacytidine, but at a reduced rate when compared to the parent line. Five subclones of the 10T1/2 line which were the progeny of single cells all expressed both the muscle and fat phenotypes following 5-azacytidine treatment. The effects of the analog are therefore not due to the selection of preexisting myoblasts or adipocytes in the cell populations. Rather, it is possible that 5-azacytidine, after incorporation into DNA, causes a reversion to a more pluripotential state from which the new phenotypes subsequently differentiate.
A bone morphogenetic protein (BMP) obtained in solution by digestion of demineralized rabbit cortical bone matrix with bacterial collagenase retains its biologically active conformation in a neutral salt/ethylene glycol mixture. BMP may be insolubilized by coprecipitation with calcium phosphate and resolubilized by chemical extraction with a neutral salt in the same solvent mixture. Upon concanavalin A-Sepharose chromatography, BMP is bound by hydrophobic interaction and carbohydrate recognition and is recovered by elution with either alpha-methyl mannoside or ethylene glycol solvent mixture. Implants of both eluates and the extracts of the coprecipitate in double-walled diffusion chambers induce transmembrane bone morphogenesis. BMP is not species specific; rabbit BMP induces new bone formation in the rat. The present observations indicate that BMP is a glycoprotein.
THE importance of collagens in growth and differentiation1,2 and in cell adhesion3 is well documented. We have previously shown that cell-free, demineralised, collagenous bone matrix induces the formation of cartilage, bone and bone marrow4-6. One of the early responses to implanted collagenous matrix is the appearance of connective tissue mesenchymal cells adjacent to the implanted matrix before their differentiation into cartilage. We present here direct evidence that collagenous bone matrix is a local mitogen for connective tissue cells. We find that the collagenous bone matrix is a potent inducer of ornithine decarboxylase (EC 4.1.1.17, ODC), an enzyme involved in polyamine biosynthesis7 and considered as an early marker of cell proliferation8. These studies have been corroborated by incorporation of 3H-thymidine into acid-precipitable material and by autoradiography. Our results reveal the local influence of insoluble collagenous bone matrix on proliferation of mesenchymal cells and imply a role for this extracellular matrix in anchorage-dependent9 events in cell growth and differentiation.
A soluble bone morphogenetic protein (BMP) along with other noncollage-nous proteins including only a relatively insignificant quantity of hydroxyproline-con-taining peptides were extracted from rabbit cortical bone by the action of an aqueous-nonaqueous solvent mixture. As it is bound by carbohydrate recognition and hydropho-bic interaction with Con A, BMP has an essential characteristic of a glycoprotein. When α-methyl mannoside and ethylene gly-col elutes of proteins bound to a Con-A column are implanted inside of double-walled diffusion chambers, deposits of cartilage and bone develop on the outside. While estimates of the quantity of BMP are less than 0.01% of the dry weight of bone matrix, it is possible to concentrate and purify a biologically active BMP by application of the principles of hy-drophobic protein chemistry.
The results of previous reports demonstrated that osteoblasts develop in cultures derived from phenotypically unexpressive stage 24 chick limb mesenchymal cells. The observations reported here suggest that initial cell plating densities may provide environmental conditions deterministic to a particular limb phenotype. Quantitative microscopic studies, histochemical localization of calcium phosphate, and electron microscopy indicate that osteoblasts develop in cultures derived from stage 24 limb mesenchymal cells. Additionally, 1–3% of the cells from stage 24 limbs are associated with mineral deposits when plated at initial high densities (5 × 106 cells per 35-mm culture dish), while more than 50% of the cells are associated with cartilage by Day 9. Cultures plated at intermediate seeding densities (between 2.0 and 2.5 × 106 cells per 35-mm culture dish) have minimal cartilage development, and approximately 20% of the cells are associated with mineral by Day 9. Furthermore, cultures prepared from stage 31 limb mesenchymal cells form well-developed bone nodules with both osteoblasts and osteocytes present, but no cartilage. It is clear from these observations and from a consideration of the initiation of osteogenesisin vivo that the initiation of bone development in the limb is not associated with cartilage development. Based on these studies and observations on the effect of nutrient factors on phenotypic expression in culture, an hypothesis is presented relating differential vascularization and nutrient flow to the determination of limb phenotypesin vivo.
Freshly excised embryonic rat skeletal muscle has been shown to form hyaline cartilage when organ cultured upon demineralized rat bone (bone matrix). Since skeletal muscle is composed of fibrous connective tissue (C.T.) as well as muscle cells, the cartilage could arise from either of these sources. The object of this study was to determine whether cartilage arose from fibrous connective tissue or muscle cells, or both, and whether the ability to form cartilage is limited to tissues derived from somatic mesoderm. Control experiments demonstrated that 19-day embryonic rat skeletal muscle formed cartilage when organ cultured on bone matrix after dissociation and cultivation in vitro, and that 11-day embryonic chick muscle also formed cartilage, although less reproducibly (3 out of 10 cases). Fibroblasts and skeletal muscle were cloned from similar suspensions of dissociated muscle in order to test these purified cell types. Dermis, vascular tissue, and tendons were mechanically removed prior to dissociation in order to eliminate fibroblasts from contaminant sources. Cloned fibroblasts, derived from rat skeletal muscle, formed cartilage in three out of three cases. It was not possible to clone sufficient rat skeletal muscle to place an aggregate onto bone matrix. An aggregate of several hundred chick skeletal muscle clones formed cartilage on bone matrix. The freshly excised C.T. capsules of embryonic chick thyroid and lung were tested for the ability to form cartilage as nonskeletal C.T. derivatives. The epithelial rudiments of thyroid and lung were also tested as endodermal derivatives. Chick cornea was similarly tested as an ectodermal derivative. Of these tissues, only the C.T. capsules formed cartilage. The results demonstrate that various C.T. cell types may alter their phenotype well after that stage at which their differentiation is thought to be stabilized, and that the ability to differentiate as cartilage may be common to all C.T. cells. The option of differentiating along a certain variety of pathways may depend more upon local conditions than on a predetermined pattern.
The bone inducing factor derived from BF osteosarcoma was purified in the following manner. Step 1. The sarcoma, grown in CBA mice, was excised and lyophilized. Step 2. The powder was washed with chilled acetone. Step 3. The acetone-treated powder was then homogenized with chilled distilled water. Step 4. Washing with 0.15M KCl. Step 5. The precipitate was incubated in in 0.2 N NH2OH, pH7.0, for 48 H at 25 degrees. After Step 5, the bone-forming activity showed a slight increase; however, the factor remained insoluble. The properties of the factor were as follows. The factor is relatively relatively heat stable; the osteogenic activity survived the treatment at 75 degrees for 15 min or at 55 degrees for 19 h. The activity was easily lost by mechanical shaking. Incubation with DNase, RNase, neuraminidase, chondroitinase ABC and beta-galactosidase left the osteogenic activity intact, but treatment with either pronase or collagnease destroyed this activity. The results suggest that the factor may be a protein. The activity was seen with the lyophilized BF osteosarcoma cells (without matrix), and it is probable that the factor was exclusively synthesized in the cells. The bone formation, observed across a millipore filter when living BF osteosarcoma enclosed in a millipore chamber was implanted in mice, suggests the synthesis and secretion of the factor from the cells.
A direct approach has been developed for quantitative identification of the calcium-binding amino acid, gamma-carboxyglutamate, in proteins. This should be advantageous for the study of numerous systems where specific roles for the binding of calcium or other divalent cations are suspected. Investigation of mineralized tissue, where calcium-binding proteins are implicated in the mineralization process, revealed that gamma-carboxyglutamate was present in proteins solubilized from chicken bone with neutral aqueous ethylenediamine tetraacetic acid. This was established by direct isolation of the amino acid from alkaline hydrolysates and its quantitative conversion to glutamic acid by decarboxylation in 0.05 M HCl at 100 degrees. The kinetics of decarboxylation and chromatographic behavior are identical to those of gamma-carboxyglutamate from human prothrombin. After resolution of the soluble bone proteins by phosphate gradient elution from hydroxyapatite, gamma-carboxyglutamate was found to be concentrated primarily in one BaSO4-adsorbable anionic protein species; bone collagen was devoid of the amino acid. In view of the recently discovered requirement of vitamin K for generation of calcium binding sites (gamma-carboxyglutamate) by gamma-carboxylation of specific glutamic acid residues in prothrombin, our findings may implicate vitamin K metabolism in normal bone development and suggest a role for the gamma-carboxyglutamate-rich protein in regulation of calcium salt deposition in mineralized tissues.
Beta-Mercaptoethanol (beta-ME) or dithiothreitol (DTT) reduction extinguishes the capacity of bone matrix gelatin to produce new bone following implantation in a muscle pouch. If the reducing solution is used in concentrations of 50 mmoles/l or less, the extinction can be partially reversed by bubbling of oxygen through the solution for one hour. Sulfhydryl group blocking reagents prevent reoxidation of reduced bone gelatin, and restoration of the bone morphogenetic property (BMP). Unspecific borohydride reduction at 37 degrees destroys bone yield irreversibly, but at 2 degrees reduction of free aldehyde groups in bone gelatin does not prevent beta-ME and DTT reversible extinction. These observations are interpreted to suggest that the disulphide linkage may be an essential part of the biologically active conformation of either a non-collagenous bone morphogenetic polypeptide firmly bound to collagen or a collagen by-product entrapped within a water insoluble gel matrix.
The bone morphogenetic property of bone matrix is degraded at 25 ° to 37 °C within 24 hours after a bone is removed from the body. The degradation occurs in the intact undemineralized bone from the action of endogenous enzymes, presumably neutral proteinases at pH optima of 7 · 0 to 7 · 4. Degradation is: more rapid at physiologic than at acid pH; heat inactivated in the range between 40 ° and 60 °C; slow at 2 °C over a period of 7 days in EDTA at pH 7 · 4. Degradation is inhibited by iodoacetic acid at concentrations as low as 3 · 0 mmoles per liter either in phosphate buffer or EDTA. Degradative activity of endogenous enzymes, as measured by the yield of bone from implants of matrix, is comparable to those obtained from matrix treated with trypsin at 15 °C, pH 7 · 6 over a period of 12 hours. These enzymes include a neutral proteinase (BMP-ase) which degrades bone morphogenetic protein (BMP) without mobilizing bone collagen hydroxy-proline as rapidly and as selectively as a specific functional entity. Observations on carboxypeptidase A and thermolysin cleavage of phenylalanine groups and data on acetylation of tyrosyl groups reducing bone yield suggest aromatic amino acids may be necessary for the biologically active conformation of BMP.
EXPERIMENTS by Charles B. Huggins and his associates,1 and many others during the past thirty years,2-11 demonstrate that whole toothbuds and parts of toothbuds possess the capacity to grow and develop in areas of the body other than the jawbone, even in the form of explants in tissue culture. The toothbud not only continues its characteristic development, but also exhibits the capacity to induce undifferentiated connective tissues to form bone. To explain these observations, Hoffman12 postulates that the enamel organ and its derivative, Hertwig's epithelial root sheath, stimulates connective tissue cells to differentiate into bone cells, while Zussman13 contends that odontoblasts which had previously participated in dentin formation can produce bone even when not in contact with oral tissues. Except that the new bone originates from proliferating living cells in both the transplanted tooth and in the host bed, present knowledge of the local physiology of
The fate of implants of decalcified dentine was compared with that of decalcified bone, tendon and muscle in three different sites: muscle pouch, drill-hole in mandible, and extracted tooth socket. Within the implants of dentine and bone matrix in areas undergoing resorption by collagenolytic mesenchymal cells and sprouting capillaries, a consistently reproducible induction system was set up for osteogenesis. Similarly prepared tendon and muscle implants did not induce osteogenesis. Except that the new bone developed from the interaction of hypertrophied mesenchymal cells with extracellular substances in the matrix of dentine and bone, and that a film of cement substance generally separates the old and new tissue, the local physical-chemical reactions are obscure. Transfer of 3H-glycine does not appear to occur locally between the old dentine matrix and ingrowing new cells of the host.
Insoluble bone gelatin with inclusions of insoluble noncollagenous protein produces new bone when implanted in muscle in allogeneic rats. The implanted residue provides the milieu for expression of bone morphogenetic potential of migratory mesenchymal cells. Neutral buffer solutions activate endogenous enzymes that degrade components essential for cell interactions and differentiation of bone. Chloroform-methanol either denatures or extracts constituents responsible for degradation. Insoluble bone gelatin produces new bone after extraction at 2 degrees with neutral salts, 0.5 M EDTA, 0.1 M Tris.HCl, 4 M urea, 0.5 M hydroxylamine, and 10 M KCNS, as well as after limited digestion with pepsin or collagenase, but not after extraction with 5 M guanidine, 7 M urea, water saturated with phenol, or after alkali hydrolysis with 0.1 N NaOH. The specific activity of cell populations interacting with insoluble bone gelatin suggests that a chemical bond between collagen and a noncollagenous protein or part of a protein, cleaved by a neutral proteinase, controls the bone morphogenetic reaction.
Bone matrix demineralized in 0.6 N HCl at 2° for 24 h and implanted in muscle in allogeneic rats possesses consistently reproducible bone morphogenetic activity. Experiments on implants of matrix, obtained from donors injected with3H-tyrosine or3H-tryptophan, or Na35SO4, suggest that bone morphogenetic property is a protein or apart of a protein that is (1) insoluble in buffer solutions, pH 3.6 and 5.0; (2) degraded in buffer solutions at pH 7.4 by an endogenous sulfhydryl-group neutral proteinase; (3) digested by trypsin at 15° within 8 h without solubilization of the helical regions, possibly even without degradation of the nonhelical ends of the bone collagen molecule, and without any loss of the periodic ultrastructure of the collagen fibrils; (4) degraded or removed by 0.1 N NaOH at 2° within 24 h without solubilization of collagen; (5) biologically active even after nitration of tyrosyl groups with tetranitromethane. The release of only one-third of the radioactivity with loss of nearly all yield of new bone by limited tryptic digestion of3H-borohydride-reduced matrix indicates that the bone morphogenetic response is the function of a non-collagenous component. Autoradiographs of implants of matrix with non-collagenous proteins labelled with3H-tryptophan,3H-tyrosine, or both3H-tyrosine and3H-phenyl-alanine demonstrate random dissemination of the radioactive constituents and no evidence of local transfer of labelled proteins or soluble protein derivatives. Hypothetically, the bone morphogenetic response is controlled by an insoluble acidic bone morphogenetic protein or polypeptide (BMP) and a soluble neutral proteinase (BMP-ase) resembling trypsin in activity except functionally more specific for BMP. Firmly bound but separable from bone collagen, BMP is one of many short-lived morphogenetic substances appearing and disappearing throughout embryonic development and persisting in postfetal life. Where the BMP receptor resides and how it activates cell mechanisms of differential repression and derepression of such genes as code for osteogenesis is unknown.
Optimal amounts of demineralized bone matrix possess the ability to coagulate platelet-free heparinized, citrated, and oxalated blood plasmas of guinea pigs. Clotting constituents become denatured in contact with the insoluble coagulant proteins. Quantities in excess of optimal modify plasma so that it does not gel when thrombin is added. The newly described coagulant effects are not restricted to the bone matrix, but are present also in the demineralized matrices of tooth and ivory, and in denatured tendon as well. They are regulated properties that were not demonstrated in mineralized bone or native tendon. The coagulant attributes of bone matrix are consistent with those of electropositive polymers of a specific sort.
Brief exposure of rat bone matrix to highly charged electrochemicals had profound but reversible effects on its competence to transform fibroblasts into chondroblasts and osteoblasts. Suppression and subsequent reactivation of this function were influenced critically by the charge of the electrochemical reagent and the pH of the reaction mixture. In dilute acids, Evans Blue, a long electronegative molecule, suppressed transforming competence, whereas hexadimethrine, a polycationic quaternary ammonium base, failed to do so. A cycle of inactivation-restoration of transforming competence was achieved by sequential treatment of bone matrix with Evans Blue followed by hexadimethrine; an electrochemical complex was demonstrated by histochemistry. In dilute alkalies, hexadimethrine inhibited, whereas, Evans Blue did not suppress the transforming function of the bone matrix. Electric charge characteristics on the surface of the bone matrix are of crucial significance for the fibroblast-transforming attribute.
Upon a substratum of insoluble bone matrix gelatin (BMG) in tissue culture, mesenchymal cells migrate out of muscle, proliferate, reaggregate on the second day and develop into cartilage on the eighth day. Mesenchymal cells proliferating in culture media containing BrdU during the first 2-3 days are irreversibly inhibited from differentiation and development of cartilage. In media containing BrdU during any interval between 3 and 8 days of culture, inhibition is reversible and cartilage develops almost invariably. Hence acquisition and stabilization of the differentiated state occurs in less than 3 days and within the 24 hr interval between 2 and 3 days coinciding with the time of interaction of mesenchymal cell filopods with surface substances of BMG.
Wandering histiocytes, foreign body giant cells, and inflammatory connective-tissue cells are stimulated by degradation products
of dead matrix to grow in and repopulate the area of an implant of decalcified bone. Histiocytes are more numerous than any
other cell form and may transfer collagenolytic activity to the substrate to cause dissolution of the matrix. The process
is followed immediately by new-bone formation by autoinduction in which both the inductor cells and the induced cells are
derived from ingrowing cells of the host bed. The inductor cell is a descendant of a wandering histiocyte; the induced cell
is a fixed histiocyte or perivascular young connective-tissue cell. Differentiation of the osteoprogenitor cell is elicited
by local alterations in cell metabolic cycles that are as yet uncharacterized.
A simple order-of-magnitude calculation suggests that diffusion may be
the underlying mechanism in establishing morphogenetic gradients in
embryonic development.
The nucleoside analog 5-azacytidine (5-aza-CR) induced marked changes in the differentiated state of cultured mouse embryo cells and also inhibited the methylation of newly synthesized DNA. The DNA strand containing 5-aza-CR remained undermethylated in the round of DNA synthesis following analog incorporation. The extent of inhibition of DNA modification and induction of muscle cells in treated cultures were dependent on the 5-aza-CR concentration over a narrow dose range. Experiments with the restriction enzyme Hpa II, which is sensitive to cytosine methylation in the sequence CCGG, demonstrated that the DNA synthesized in 5-aza-CR-treated cultures was maximally undermethylated 48 hr after treatment. Three other analogs of cytidine, containing a modification in the 5 position of the pyrimidine ring [5-aza-2'-deoxycytidine(5-aza-CdR), pseudoisocytidine (psi ICR) and 5-fluoro-2'-deoxycytidine(FCdR)] also induced the formation of muscle cells and inhibited DNA methylation. In contrast, 1-beta-D-arabinofuranosylcytosine (araC) and 6-azacytidine (6-aza-CR) did not inhibit DNA methylation or induce muscle formation, whereas 5-6-dihydro-5-azacytidine (dH-aza-CR) was a poor inducer of muscle cells and a poor inhibitor of DNA methylation. These results provide experimental evidence for a role for DNA modification in differentiation, and suggest that cytidine analogs containing an altered 5 position perturb previously established methylation patterns to yield new cellular phenotypes.
The roles of cartilage-derived factor and bone-derived growth factor on skeletal growth were evaluated by comparing their effects on cultured chondrocytes with those of multiplication-stimulating activity and mitogenic polypeptides isolated from pituitary glands and a fibrosarcoma. Fibroblast growth factor and DNA synthesis factor derived from a Rhodamine fibrosarcoma increased the incorporation of [3H]thymidine into chondrocytes in a dose-dependent manner without either stimulation or inhibition of proteoglycan synthesis. The extent of stimulation of DNA synthesis by fibroblast growth factor and DNA synthesis factor were much greater than those of multiplication-stimulating activity, cartilage-derived factor and bone-derived growth factor. On the contrary, multiplication-stimulating activity, cartilage-derived factor and bone-derived growth factor caused stimulation of proteoglycan synthesis by chondrocytes, measured as incorporation of 35SO42− into material precipitated with cetylpyridinium chloride. This converse relationship between DNA synthesis and proteoglycan synthesis suggests a specific mechanism by which somatomedin and somatomedin-like factors enhance proteoglycan synthesis in chondrocytes. In addition, fibroblast growth factor and DNA synthesis factor had additive effects with multiplication-stimulating activity in increasing incorporation of [3H]thymidine, but did not affect multiplication-stimulating activity-induced increase in proteoglycan synthesis. Thus, DNA synthesis factor present in a Rhodamine fibrosarcoma seems to be a fibroblast growth factor-like mitogenic factor rather than a multiplication-stimulating activity-like factor, although a human fibrosarcoma cell line has been shown to produce multiplication-stimulating activity-related peptides (De Larco, J.E. and Todaro G.J. (1978) Nature, 272, 356–358.)
In the first paper in this series (Nathanson, M. A., and Hay, E. D. (1980). Develop. Biol. 78, 301–331), we described the ultrastructural alterations that take place when embryonic skeletal muscle is induced to form hyaline cartilage by demineralized bone matrix in vitro. In this paper, we analyze the pattern of appearance of chondroitin sulfates and dermatan sulfate in injured muscle in situ and in explants of muscle cultured either on bone matrix or on collagen gel. We also investigate the effects of exogenous glycosaminoglycans on the cultures to determine whether chondroitin sulfate (Ch-S) and hyaluronic acid (HA) can enhance or inhibit the biochemical differentiation of cartilage under these conditions. Our results indicate that during the first morphological phase, 1–3 days in vitro, there is an increased sulfate uptake, a shift in the relative abundance of Ch-S, and an increase in the ratio of chondroitin-4-sulfate (Ch-4-S) to chondroitin-6-sulfate (Ch-6-S); this change is correlated with the transformation of myoblasts to fibroblast-like cells in both types of cultures. A similar increase in the ratio occurs in injured muscle in situ, suggesting that phase I is a regenerative response. Explants on bone matrix sustain Ch-4-S levels between 4 and 5 days (phase II) and show a large increase in Ch-4-S and sulfate incorporation when they form cartilage at 6–10 days (phase III). Explants on collagen gels regenerate muscle at 4–10 days with decreasing ratios and decreasing sulfate incorporation. The data demonstrate that an environmental influence, such as trauma, is sufficient to alter the biosynthetic expression of skeletal muscle and that under appropriate conditions (such as the presence of bone matrix) this response may be augmented, leading to the synthesis of extracellular matrix components at ratios characteristic of cartilage. Exogenous Ch-S and HA did not significantly effect this overall pattern. These results are discussed in relation to the morphological observations presented in the preceding paper.
To evaluate the requirement of vascular and/or neurotrophic-derived factors on determination and differentiation of chondrogenic
and osteogenic phenotypes, early embryonic quail and mouse mandibular processes were cultured using a modified Trowell method
in a serumless, chemically-defined medium for 10 days. Quail HH stage 22 and mouse Theiler stage 16 mandibular processes formed
cartilage and produced osteoid under these experimental conditions. Chondrogenic and osteogenic phenotypes were expressed
without serum or other exogenous growth-promoting influences.
Human cortical bones were extracted with EDTA, and the residue after EDTA extraction was digested with bacterial collagenase. Ten plasma proteins were identified and quantitated in the EDTA extracts. Three of them--IgE, IgD, and alpha 1acid-glycoprotein--had not previously been described in bone or dentine. Five plasma proteins identified in collagenase digests are albumin, IgG, IgA, IgE, and alpha 1acid-glycoprotein. IgE, alpha 1acid-glycoprotein, and alpha 2HS-glycoprotein were found to be concentrated in the bone more than other plasma proteins by factors between 11 and 525. The identification of plasma proteins was facilitated by the addition of polyethylene glycol in agarose gel. The presence of plasma proteins both in EDTA extracts and in collagenase digests suggests their structural role in bone.
Human bone morphogenetic protein (hBMP) was chemically extracted from demineralized gelatinized cortical bone matrix by means of a CaCl2 X urea inorganic-organic solvent mixture, differential precipitation in guanidine hydrochloride, and preparative gel electrophoresis. hBMP is isolated in quantities of 1 mg/kg of wet weight of fresh bone, and has the amino-acid composition of an acidic polypeptide. The mol wt is 17 to 18 k-Da (kilodaltons). Implants of the isolated 17-kDa protein are very rapidly adsorbed and produce a smaller volume of bone than protein fractions consisting of 24-, 17-, and 14-kDa proteins. Since the isolated 24- and 14-kDA components lack hBMP activity, the kinetics of the bone morphogenetic processes including the function of other proteins as carrier molecules, await investigation.
Embryonic chick bones growing in organ culture release a substance into the culture medium which stimulates bone formation in previously untreated bones. This “conditioned” medium also enhances proliferation of monolayer cultures of chick calvaria cells in serum-free medium. The active principle is nondialyzable, indicating a molecular weight greater than 12,000 daltons. Dialysis also separates the mitogenic activity from a low-molecular-weight inhibitor. The amount of the mitogen found in conditioned medium increases as the rate of bone resorption increases in response to treatment with parathyroid hormone or 1,25-dihydroxyvitamin D3. Maximal stimulation of DNA synthesis in calvaria cells is evident with conditioned medium obtained 3 to 5 days after treatment of bone cultures with parathyroid hormone. The cells must be treated with the conditioned medium continuously for 20 hr in order to obtain peak enhancement of DNA synthesis; there is no detectable effect in the first 8 hr. In contrast, the inhibitor acts within 4 hr. The data suggest that the stimulatory factor acts to increase cell proliferation by promoting entry of cells into the S phase of mitosis. We conclude that this stimulator is a locally produced regulator of bone formation, probably acting via an increased proliferation in osteoblast precursor cells.
Subcutaneous implantation of demineralized diaphyseal bone matrix in allogeneic rats results in the local induction of endochondral bone differentiation. We have explored the potential of three dissociative extractants, 4 M guanidine hydrochloride (Gdn . HCl), 8 M urea/1 M NaCl, and 1% NaDodSO4 at pH 7.4, containing protease inhibitors to solubilize putative inductive molecules in the bone matrix. Extraction of bone matrix with any one of these extracts resulted in the loss of the bone inductive property. The solubilized extracts were then reconstituted with the residue by dialysis against water. The various reconstituted matrices were bioassayed for bone inductive potential by quantitation of alkaline phosphatase activity and 45Ca incorporation on day 12 after implantation. There was complete recovery of biological activity after reconstitution of the residues with each of the three extracts. Polyacrylamide gel electrophoresis of the extracts revealed similar protein profiles. Gel filtration of the 4 M Gdn. HCl extract on Sepharose CL-4B showed a heterogeneous broad peak. When fractions of that peak containing proteins less than 50,000 daltons were reconstituted with inactive 4 M Gdn . HCl-treated bone matrix and then implanted, new bone was induced. These observations demonstrate the dissociative extraction and successful biological reconstitution of bone inductive macromolecules in demineralized bone matrix.
Osteonectin is a 32,000 dalton bone-specific protein that binds selectively to both hydroxyapatite and collagen. When osteonectin is bound to insolubilized type I collagen, the resultant complex binds synthetic apatite crystals and free calcium ions. The osteonectin-collagen complexes also nucleate mineral phase deposition from metastable balanced salt solutions, Antibodies to osteonectin cross-react with bone and, to a lesser extent, dentin, but not with other tissues. The protein is localized to mineralized bone trabeculae and occurs at higher levels in the matrix than in the cells of bone. These studies suggest that osteonectin is a tissue-specific protein, linking the bone mineral and collagen phases, perhaps initiating active mineralization in normal skeletal tissue.
Trephine defects in the adult rat skull 0.8 cm in diameter, which do not spontaneously heal, were filled with a bovine bone morphogenetic protein (BMP) fraction. The defects healed not only by bony ingrowth from the trephine rim, but also by proliferation of pervascular mesenchymal-type cells (pericytes) of the dura mater. Under the influence of BMP, dural pericytes differentiated into chondroid and woven bone. Between three and four weeks postimplantation, sinusoids formed and the woven bone remodelled into lamellar bone. Concurrently, blood-borne bone marrow cells colonized the bone deposits, and the diploe were restored. Demonstrating that it is soluble in interstitial fluid, and diffusible across a nucleopore membrane (which isolated the bony margins of the skull), BMP induced new bone formation in the underlying dura and complete repair of the defect. The response of the dura to the BMP fraction produced more new bone than the response to allogeneic bone matrix. The BMP-induced repair was dose dependent; the quantity of new bone was proportional to the dose of the implanted BMP.
The preceding paper [Farley, J. R., & Baylink, D. J. (1982) Biochemistry (preceding paper in this issue)] detailed the purification of a potent growth factor from human bone matrix. The purified protein (Mr 83 000) was shown to increase, in vitro, both the proliferation rate of embryonic chick calvarial cells and the growth rate of embryonic chick bone. We now report that the effect of this human skeletal growth factor on chick calvarial cell proliferation in vitro is saturable, prolonged, and inductive. The effect was saturable in that dose/response studies revealed a Kmapp of 12.5 nM/103 cells in a 1-mL volume. The absolute value of Kmapp varied with initial cell density. Although the peak response for DNA synthesis occurred after 16-22 h of exposure to the purified factor (508% of control, p < 0.001), DNA synthesis was still elevated (280% of control, p < 0.001) after 66 h of continuous exposure. The peak response seen after 16-22 h was evident without the continuous presence of the factor (i.e., when cells preincubated with the factor for 5-6 h were incubated in factor-free medium), suggesting that the effect is inductive. Actual counts of calvarial cells in culture revealed that in response to added factor, both cell number and maximum cell density (161% of controls, p < 0.001) were increased. This factor was also shown to stimulate chick calvarial cell proliferation in confluent cultures (185% of control, p < 0.001). The effect on calvarial cell proliferation in subconfluent cultures was not sensitive to indomethacin, but was subject to competitive inhibition by isobutylmethylxanthine and noncompetitive inhibition by trifluoperazine (a calmodulin antagonist). The factor also increased the proliferation rate of human bone cells in vitro, to as much as 1090% of serum-free controls. Preliminary studies suggest that the concentration of the factor in trabecular bone is greater than the concentration in cortical bone (p < 0.001).
The bone morphogenetic property in bone matrix, recently demonstrated to be a glycoprotein, and the mesenchymal cell response to it, are growth hormone dependent. Both can be partially restored by the administration of bGH to hypophysectomized rats.
Previous studies have demonstrated that embryonic skeletal muscle is competent to form hyaline cartilage when cultured in vitro on demineralized bone matrix (Nogami, H., and Urist, M. R. (1970). Exp. Cell Res.63, 404–410; Nathanson, M. A., et al. (1978). Develop. Biol.64, 99–117). The present experiments were undertaken to determine the nature of the morphological alterations which attend this phenotypic transformation and to investigate the ultrastructural characteristics of the myoblasts and fibroblasts of skeletal muscle during the transformation. Nineteen-day embryonic rat limb muscles were minced and the tissue fragments explanted to bone matrix or collagen gels. The trauma of excision and mincing causes syncytial myotubes to degenerate and the nuclei of mononucleate cells to enter a heterochromatic “resting stage.” In culture, nuclei of mononucleate cells rapidly regain euchromasia. No myoblast or fibroblast cell death can be detected. On bone matrix, the entire mononucleate population transforms into fibroblast-like cells. Myoblasts are the major contributor to this population; they dissociate from the degenerate myotubes and begin to acquire endoplasmic reticulum by 24 h in vitro. The fibroblast-like morphology persists through 4 days in vitro. By 6 days in vitro some of these fibroblast-like cells acquire the phenotypic characteristics of chondrocytes, and by 10 days masses of hyaline cartilage are found. In control explants of skeletal muscle onto collagen gels, the heterochromatic nuclei of the mononucleated cells expand after 24 hr in vitro, but the mononucleated cells remain as myoblasts and fibroblasts and begin to regenerate skeletal muscle by 4 days in vitro. No cartilage forms. The results indicate that both myoblasts and fibroblasts have chondrogenic potential when grown on demineralized bone. It is tempting to conclude that the embryonic mesenchymal cells which give rise to skeletal muscle, cartilage, and other connective tissue of the limb have similar developmental potentials and that local influences, rather than separate cell lineages, account for the final pattern of differentiation.