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The dependence of autologous chondrocyte transplantation on varying cellular passage, yield and culture duration
... Finally, optimal graft production may affect the final transplant outcome with optimized joint homeostasis. In a previously published study, it could be shown that cell culture parameters are able to strongly impact transplant performance in vitro and in vivo [17]. Here, we could show that such change in culture conditions also affected pro-inflammatory cytokines and matrix metalloproteinases in a significant manner. ...
... In contrast, Gavenis et al. showed no improvement by a prolonged membrane-holding time [31]. Today, it is known that a short membrane-holding time has beneficial effects on graft maturation and differentiation [17]. This results in matrix deposition surrounding the cells, which allows the transmission of biomechanical signals through the matrix rather than onto the naked cell. ...
... Within the procedures involving animal care and treatment, all efforts were made to minimize suffering. The here-presented research draws from the study protocol published in the work of Salzmann et al. [17], but seeks to investigate a completely new hypothesis. ...
Cartilage defects represent an increasing pathology among active individuals that affects the ability to contribute to sports and daily life. Cell therapy, such as autologous chondrocyte implantation (ACI), is a widespread option to treat larger cartilage defects still lacking standardization of in vitro cell culture parameters. We hypothesize that mRNA expression of cytokines and proteases before and after ACI is influenced by in vitro parameters: cell-passage, cell-density and membrane-holding time. Knee joint articular chondrocytes, harvested from rabbits (n = 60), were cultured/processed under varying conditions: after three different cell-passages (P1, P3, and P5), cells were seeded on 3D collagen matrices (approximately 25 mm 3) at three different densities (2 × 10 5 /matrix, 1 × 10 6 /matrix, and 3 × 10 6 /matrix) combined with two different membrane-holding times (5 h and two weeks) prior autologous transplantation. Those combinations resulted in 18 different in vivo experimental groups. Two defects/knee/animal were created in the trochlear groove (defect dimension: ∅ 4 mm × 2 mm). Four identical cell-seeded matrices (CSM) were assembled and grouped in two pairs: One pair giving pre-operative in vitro data (CSM-i), the other pair was implanted in vivo and harvested 12 weeks post-implantation (CSM-e). CSMs were analyzed for TNF-α, IL-1β, MMP-1, and MMP-3 via qPCR. CSM-i showed higher expression of IL-1β, MMP-1, and MMP-3 compared to CSM-e. TNF-α expression was higher in CSM-e. Linearity between CSM-i and CSM-e values was found, except for TNF-α. IL-1β expression was higher in CSM-i at higher passage and longer membrane-holding time. IL-1β expression decreased with prolonged membrane-holding time in CSM-e. For TNF-α, the reverse was true. Lower cell-passages and lower membrane-holding time resulted in stronger TNF-α expression. Prolonged membrane-holding time resulted in increased MMP levels among CSM-i and CSM-e. Cellular density was of no significant effect. We demonstrated cytokine and MMP expression levels to be directly influenced by in vitro culture settings in ACI. Linearity of expression-patterns between CSM-i and CSM-e may predict ACI regeneration outcome in vivo.
... 3 Novel techniques, such as matrix-assisted chondrocyte implantation, have been introduced to reduce cellular dedifferentiation and also improve transplant handling via three-dimensional (3-D) cell culture conditions. 4 Yet it has been shown that static, free-swelling culture does not result in a significant redifferentiation of articular chondrocytes in 3-D culture. 3,5 As mechanical stimulation is one of the major regulators known to maintain in situ cartilage integrity and function, various experimental models have shown that physiological mechanical stimulation results in chondrocyte maturation in vitro. 6 Bioreactors are capable of reproducing a joint-like surrounding by providing compression, shear, and fluid flow. ...
... 31 Moreover, supporting the fact that bioreactor tissue engineering mimicking joint movement can produce similar results as animal experiments, it was additionally reported in one previous study by Salzmann and colleagues that linear mixed regression exposed that with increasing passage the expression of differentiation targets decreased in vitro and in vivo. 5 Practical chondrocyte transplantation techniques face an inherent limitation of transplanting a tissue-engineered cell-scaffold construct containing subcultured chondrocytes with inferior quality. Autologous chondrocyte implantation techniques commonly apply passaged (passage 3-5) chondrocytes, which are placed under a water-tight periosteum or under/within a 3-D membrane. ...
... This information parallels previous reports. 5,35 Even though it has to be reminded that in general static (and non-chemical enhanced) free-swelling culture of matrixsurrounded chondrocytes does not strongly enhance chondrogenesis. 36 Albrecht and colleagues recently reported that scaffold characteristics and culture conditions highly influenced gene expression among four different industrial transplant types (MACI, HyalograftC, CaReS, and Novocart 3D), attesting that simple culture parameters may have profound impact on the tissue regeneration following matrix-associated chondrocyte transplantation. ...
To explore the effect of shifting in vitro culture conditions regarding cellular passage and onset of loading within matrix-associated bovine articular chondrocytes cultured under free-swelling and/or dynamical loading conditions on general chondrocyte maturation.
Primary or passage 3 bovine chondrocytes were seeded in fibrin-polyurethane scaffolds. Constructs were cultured either free-swelling for 2 or 4 weeks, under direct mechanical loading for 2 or 4 weeks, or free swelling for 2 weeks followed by 2 weeks of loading. Samples were collected for glycosaminoglycan (GAG) quantification, mRNA expression of chondrogenic genes, immunohistochemistry, and histology.
Mechanical loading generally stimulated GAG synthesis, up-regulated chondrogenic genes, and improved the accumulation of matrix in cell-laden constructs when compared with free-swelling controls. Primary chondrocytes underwent more effective cartilage maturation when compared with passaged chondrocytes. Constructs of primary chondrocytes that were initially free-swelling followed by 2 weeks of mechanical load (delayed) had overall highest GAG with strongest responsiveness to load regarding matrix synthesis. Constructs that experienced the delayed loading regime also demonstrated most favorable chondrogenic gene expression profiles in both primary and third passage cells. Furthermore, most intense matrix staining and immunostaining of collagen type II and aggrecan were visualized in these constructs.
Primary chondrocytes were more effective than passage 3 chondrocytes when chondrogenesis was concerned. The most efficient chondrogenesis resulted from primary articular chondrocytes, which were initially free-swelling followed by a standardized loading protocol.
... [19][20][21][22][23] In the latter case, the use of matrixassisted autologous chondrocyte transplantation has become increasingly attractive since 1998, because articular cartilage defects of varying degrees can be replenished more easily and in a more targeted manner. 22,24 So far, a variety of different synthetic and natural materials have been investigated with respect to cartilage repair. 3,25 The main focus lies on restoring the complex structure and properties of hyaline cartilage to achieve long-term improvements in cartilage engineering. ...
... In the second step, the maleimide-functionalized nano particles react with the thiolated IGF-1 to form a stable chemical bond between IGF-1 and particles. 30 Therefore, 250 µL of sicastar ® -redF (50 mg/mL) was suspended in 24 amine-tosulfhydryl crosslinkers with soluble PEG spacer arms; Thermo Fisher Scientific, Waltham, MA, USA). After shaking the suspension for 2 hours at room temperature, the particles were washed twice with PBS buffer (pH =7.4, ...
Purpose
In the present study, silica nanoparticles (sNP) coupled with insulin-like growth factor 1 (IGF-1) were loaded on a collagen-based scaffold intended for cartilage repair, and the influence on the viability, proliferation, and differentiation potential of human primary articular chondrocytes was examined.
Methods
Human chondrocytes were isolated from the hyaline cartilage of patients (n=4, female, mean age: 73±5.1 years) undergoing primary total knee joint replacement. Cells were dedifferentiated and then cultivated on a bioresorbable collagen matrix supplemented with fluorescent sNP coupled with IGF-1 (sNP–IGF-1). After 3, 7, and 14 days of cultivation, cell viability and integrity into the collagen scaffold as well as metabolic cell activity and synthesis rate of matrix proteins (collagen type I and II) were analyzed.
Results
The number of vital cells increased over 14 days of cultivation, and the cells were able to infiltrate the collagen matrix (up to 120 μm by day 7). Chondrocytes cultured on the collagen scaffold supplemented with sNP–IGF-1 showed an increase in metabolic activity (5.98-fold), and reduced collagen type I (1.58-fold), but significantly increased collagen type II expression levels (1.53-fold; P=0.02) after 7 days of cultivation compared to 3 days. In contrast, chondrocytes grown in a monolayer on plastic supplemented with sNP-IGF-1 had significantly lower metabolic activity (1.32-fold; P=0.007), a consistent amount of collagen type I, and significantly reduced collagen type II protein expression (1.86-fold; P=0.001) after 7 days compared to 3 days.
Conclusion
Collagen-based scaffolds enriched with growth factors, such as IGF-1 coupled to nanoparticles, represent an improved therapeutic intervention for the targeted and controlled treatment of articular cartilage lesions.
... The effects of EGF dilutions(1,5,10,25, 50, 100, 200 ng/mL) in combination with BA (100, 300, 500 µg/mL) on cell viability were analyzed by MTT assay after 24, 48 and 72 h of treatment on (a) L929, (c) Saos-2 and (c) hAD-MSCs (mean ± standard deviation, n=3). The DMEM medium containing no compounds was used as a control. ...
Objectives
In this study, we aimed to determine the bioefficacy of epidermal growth factor (EGF), boric acid (BA), and their combination on cartilage injury in rats.
Materials and methods
In in vitro setting, the cytotoxic effects of BA, EGF, and their combinations using mouse fibroblast cell (L929), human bone osteosarcoma cell (Saos-2), and human adipose derived mesenchymal stem cells (hAD-MSCs) were determined by applying MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] test. In in vivo setting, 72 rats were randomly divided into four groups. A standard chondral defect was created and microfracture was performed in all groups. Group A was determined as the control group. In addition to the standard procedure, Group B received 100 ng/mL of EGF, Group C received a combination of 100 ng/mL of EGF and 10 µg/mL of BA combination, and Group D 20 µg/mL of BA.
Results
The cytotoxic effect of the combinations of EGF dilutions (1, 5, 10, 25, 50, 100, 200 ng/mL) with BA (100, 300, 500 µg/mL) was observed only in the 72-h application period and in Saos-2. The cytotoxic effect of BA was reduced when combined with EGF. There was no significant difference in the histopathological scores among the groups (p=0.13).
Conclusion
Our study showed that EGF and low-dose BA application had a positive effect on cartilage healing in rats. Significant decreases in recovery scores were observed in the other groups. The combination of EGF and BA promoted osteoblast growth. Detection of lytic lesions in the group treated with 20 µg/mL of BA indicates that BA may have a cytotoxic effect.
... Owing to the avascularity of cartilage and low metabolic activity of chondrocytes, such cartilage defects have limited capacity to heal spontaneously [5,6]. Several therapeutic strategies have been developed to treat cartilage damage, including bone marrow stimulation (such as microfracture and subchondral bone drilling) [7,8], autologous chondrocyte implantation [9][10][11] and autografts transplantation [12,13]. However, the complete repair of hyaline cartilage remains a major challenge in clinical practice owing to the inferior quality of regenerative tissue [14,15]. ...
Background
Cartilage damage is a common medical issue in clinical practice. Complete cartilage repair remains a significant challenge owing to the inferior quality of regenerative tissue. Safe and non-invasive magnetic therapy combined with tissue engineering to repair cartilage may be a promising breakthrough.
Methods
In this study, a composite scaffold made of Hydroxyapatite-Collagen type-I (HAC) and PLGA-PEG-PLGA thermogel was produced to match the cartilage and subchondral layers in osteochondral defects, respectively. Bone marrow mesenchymal stem cells (BMSC) encapsulated in the thermogel were stimulated by an electromagnetic field (EMF). Effect of EMF on the proliferation and chondrogenic differentiation potential was evaluated in vitro. 4 mm femoral condyle defect was constructed in rabbits. The scaffolds loaded with BMSCs were implanted into the defects with or without EMF treatment. Effects of the combination treatment of the EMF and composite scaffold on rabbit osteochondral defect was detected in vivo.
Results
In vitro experiments showed that EMF could promote proliferation and chondrogenic differentiation of BMSCs partly by activating the PI3K/AKT/mTOR and Wnt1/LRP6/β-catenin signaling pathway. In vivo results further confirmed that the scaffold with EMF enhances the repair of osteochondral defects in rabbits, and, in particular, cartilage repair.
Conclusion
Hydrogel-Hydroxyapatite-Monomeric Collagen type-I scaffold with low-frequency EMF treatment has the potential to enhance osteochondral repair.
... Cartilage or joint reconstruction is still a considerable challenge due to cartilage injury, which leads to severe pain and disability of joint inflammation [1][2][3][4][5]. The increasing clinical demands has driven a large number of clinical surgical treatments, including autografts transplantation [6], autologous chondrocyte transplantation [7], and marrow stimulation such as microfracture [8] and subchondral drilling [9]. Although successful to some extent, each treatment has its own limitations. ...
Background/Objective
We seek to figure out the effect of stable and powerful mechanical microenvironment provided by Ti alloy as a part of subchondral bone scaffold on long-term cartilage regeneration.
Methods: we developed a bilayered osteochondral scaffold based on the assumption that a stiff subchondral bony compartment would provide stable mechanical support for cartilage regeneration and enhance subchondral bone regeneration. The subchondral bony compartment was prepared from 3D printed Ti alloy, and the cartilage compartment was created from a freeze-dried collagen sponge, which was reinforced by poly-lactic-co-glycolic acid (PLGA).
Results
In vitro evaluations confirmed the biocompatibility of the scaffold materials, while in vivo evaluations demonstrated that the mechanical support provided by 3D printed Ti alloy layer plays an important role in the long-term regeneration of cartilage by accelerating osteochondral formation and its integration with the adjacent host tissue in osteochondral defect model at rabbit femoral trochlea after 24 weeks.
Conclusion
Mechanical support provided by 3D printing Ti alloy promotes cartilage regeneration by promoting subchondral bone regeneration and providing mechanical support platform for cartilage synergistically.
Translational potential statement
The raw materials used in our double-layer osteochondral scaffolds are all FDA approved materials for clinical use. 3D printed titanium alloy scaffolds can promote bone regeneration and provide mechanical support for cartilage regeneration, which is very suitable for clinical scenes of osteochondral defects. In fact, we are conducting clinical trials based on our scaffolds. We believe that in the near future, the scaffold we designed and developed can be formally applied in clinical practice.
... Owing to the avascularity of cartilage and low metabolic activity of chondrocytes, such cartilage defects have limited capacity to heal spontaneously [5,6]. Several therapeutic strategies have been developed to treat cartilage damage, including bone marrow stimulation (such as microfracture and subchondral bone drilling) [7,8], autologous chondrocyte implantation [9][10][11] and autografts transplantation [12,13]. However, the complete repair of hyaline cartilage remains a major challenge in clinical practice owing to the inferior quality of regenerative tissue [14,15]. ...
Background: Cartilage damage is a common medical issue in clinical practice. Complete cartilage repair remains a significant challenge owing to the inferior quality of regenerative tissue.
Methods: In this study, a composite scaffold made of Hydroxyapatite-Collagen type-I (HAC) and PLGA-PEG-PLGA thermogel was produced to match the cartilage and subchondral layers in osteochondral defects, respectively. Bone marrow mesenchymal stem cells (BMSC) encapsulated in the thermogel were stimulated by an electromagnetic field (EMF).
Results: The scaffold was evaluated in rabbits, and biological characteristic of BMSCs were measured. Results showed that the scaffold with EMF enhances the repair of osteochondral defects in rabbits, and, in particular, cartilage repair. EMF could promote proliferation and chondrogenic differentiation of BMSCs partly by activating the PI3K/AKT/mTOR and Wnt1/LRP6/β-catenin pathway.
Conclusion: Hydrogel-Hydroxyapatite-Monomeric Collagen type-I scaffold with low-frequency EMF treatment has the potential to enhance osteochondral repair.
... The current main therapies to heal cartilage defects include autografts transplantation [7,8], autologous chondrocytes transplantation [9,10], marrow stimulation such as subchondral drilling [11] and allografts [12]. Autografts or autologous chondrocytes transplantations perform better functions for its lower immunogenicity and better biocompatibility. ...
Type II collagen (Col-II) is one of the important organic components of the cartilage extracellular matrix (ECM). Such natural material is known for its good biocompatibility, but it could not provide a good supporting environment for seed cells due to its rapid degradation and poor strength. In the present work, different contents of Col-II were incorporated into porous polyvinyl alcohol (PVA) to fabricate porous PVA/Col-II composite hydrogels for cartilage tissue engineering. The results illustrate that, after incorporation of Col-II, the elasticity modulus of the composite hydrogels firstly increases, and then decreases (under moisture state). The elasticity modulus of PVA/Col-II (at the ratio of 1:1) hydrogels reaches 11 ± 1.7 KPa, about two-fold higher than pure PVA hydrogels (4.9 ± 0.6 KPa). Meanwhile, all hydrogels exhibit relatively high water content (> 95%) and porosity (> 75%). The degradation analysis indicates that Col-II incorporation induce a high degradation ratio of the composite hydrogels. Cell culture results show PVA/Col-II hydrogels have no negative effects on cells viability and proliferation. The PVA/Col-II hydrogels may possess a potential application in the field of articular cartilage tissue engineering and regeneration.
... 7,41 Standard ACI is hampered by the requirement of 2 interventions, a high cell-producing cost, and questionable chondrocyte function as well as phenotype at the time of implantation. 14,35,46 ACI is not available in every country and represents a constant target of national regulations in countries where it is available. A possible alternative to ACI is the application of small cartilage chips that have been previously particulated or minced. ...
Background:
Chondral and osteochondral lesions are being detected with increasing frequency. For large-diameter lesions, cell-based treatment modalities are speculated to result in the best possible outcome.
Purpose:
To prospectively evaluate the 2-year clinical and radiological results after the treatment of chondral and osteochondral knee joint lesions by a single-step autologous minced cartilage procedure.
Study design:
Case series; Level of evidence, 4.
Methods:
From February 2015 to June 2016, a total of 27 consecutive patients suffering from chondral or osteochondral lesions of the knee joint were treated using a single-step autologous minced cartilage procedure. All patients underwent preoperative and postoperative magnetic resonance imaging for the collection of AMADEUS (Area Measurement and Depth and Underlying Structures) and MOCART (magnetic resonance observation of cartilage repair tissue) scores. Clinical analysis was conducted by a numeric analog scale (NAS) for pain and knee function before the intervention and at 12 and 24 months postoperatively.
Results:
A total of 12 female and 15 male patients (mean age, 28.7 years) were evaluated for a mean of 28.2 ± 3.8 months. The mean cartilage defect size encountered intraoperatively was 3.1 ± 1.6 cm2. There was a significant decrease in pain from 7.2 ± 1.9 preoperatively to 1.8 ± 1.6 (P < .001) at 2-year follow-up. Knee function improved from a mean of 7.2 ± 2.0 preoperatively to 2.1 ± 2.3 (P < .001) at 2 years after surgery. The mean preoperative AMADEUS score was 57.4 ± 21.4. Postoperatively, the mean MOCART score was 40.6 ± 21.1 at 6-month follow-up. No correlation was observed between the clinical data and the MOCART or AMADEUS scores.
Conclusion:
Overall, the findings of this study demonstrated that patients undergoing a single-step autologous minced cartilage procedure had a satisfactory outcome at 2-year follow-up. As a result, the single-step autologous minced cartilage procedure does represent a possible alternative to standard autologous chondrocyte implantation. Longer follow-up and larger cohorts are required to define the benefits of this procedure.
... Osteochondral lesions are notably complex due to the intricated structure of the tissue and are usually associated with great instability, pain, and impaired function. A variety of clinical techniques have been used to treat these injuries, including autologous chondrocyte implantation, microfracture, autograft, and mosaicplasty [2][3][4][5][6][7][8][9][10]. These methods have several limitations, including graft deterioration/ lack of integration, mechanically-insufficient cartilage formation, and donor site morbidity, leading to often insufficient patient outcomes [11][12][13]. ...
Recent developments in 3D printing (3DP) research have led to a variety of scaffold designs and techniques for osteochondral tissue engineering; however, the simultaneous incorporation of multiple types of gradients within the same construct remains a challenge. Herein, we describe the fabrication and mechanical characterization of porous poly(ε-caprolactone) (PCL) and PCL-hydroxyapatite (HA) scaffolds with incorporated vertical porosity and ceramic content gradients via a multimaterial extrusion 3DP system. Scaffolds of 0 wt% HA (PCL), 15 wt% HA (HA15), or 30 wt% HA (HA30) were fabricated with uniform composition and porosity (using 0.2 mm, 0.5 mm, or 0.9 mm on-center fiber spacing), uniform composition and gradient porosity, and gradient composition (PCL-HA15-HA30) and porosity. Micro-CT imaging and porosity analysis demonstrated the ability to incorporate both vertical porosity and pore size gradients and a ceramic gradient, which collectively recapitulate gradients found in native osteochondral tissues. Uniaxial compression testing demonstrated an inverse relationship between porosity, ϕ, and compressive modulus, E, and yield stress, σ y , for uniform porosity scaffolds, however, no differences were observed as a result of ceramic incorporation. All scaffolds demonstrated compressive moduli within the appropriate range for trabecular bone, with average moduli between 86 ± 14–220 ± 26 MPa. Uniform porosity and pore size scaffolds for all ceramic levels had compressive moduli between 205 ± 37–220 ± 26 MPa, 112 ± 13–118 ± 23 MPa, and 86 ± 14–97 ± 8 MPa respectively for porosities ranging between 14 ± 4–20 ± 6%, 36 ± 3–43 ± 4%, and 54 ± 2–57 ± 2%, with the moduli and yield stresses of low porosity scaffolds being significantly greater (p < 0.05) than those of all other groups. Single (porosity) gradient and dual (composition/porosity) gradient scaffolds demonstrated compressive properties similar (p > 0.05) to those of the highest porosity uniform scaffolds (porosity gradient scaffolds 98 ± 23–107 ± 6 MPa, and 102 ± 7 MPa for dual composition/porosity gradient scaffolds), indicating that these properties are more heavily influenced by the weakest section of the gradient. The compression data for uniform scaffolds were also readily modeled, yielding scaling laws of the form E ∼ (1 − ϕ) 1.27 and σ y ∼ (1 − ϕ) 1.37 , which demonstrated that the compressive properties evaluated in this study were well-aligned with expectations from previous literature and were readily modeled with good fidelity independent of polymer scaffold geometry and ceramic content. All uniform scaffolds were similarly deformed and recovered despite different porosities, while the large-pore sections of porosity gradient scaffolds were significantly more deformed than all other groups, indicating that porosity may not be an independent factor in determining strain recovery. Moving forward, the technique described here will serve as the template for more complex multimaterial constructs with bioactive cues that better match native tissue physiology and promote tissue regeneration. Statement of significance: This manuscript describes the fabrication and mechanical characterization of “dual” porosity/ceramic content gradient scaffolds produced via a multimaterial extrusion 3D printing system for osteochondral tissue engineering. Such scaffolds are designed to better address the simultaneous gradients in architecture and mineralization found in native osteochondral tissue. The results of this study demonstrate that this technique may serve as a template for future advances in 3D printing technology that may better address the inherent complexity in such heterogeneous tissues.
... This flexible cytoskeleton appears to be critical in maintaining constant cell volume during the 21 day culture, and encouraged redifferentiation. This phenomenon highlights the critical influences on cell fate due to the variations in seeding density and cell-matrix holding time [13,45], since they all affect initial cell morphology in a matrix. Compared to the majority of 3D culture systems, the current single cell 3D culture eliminates cell-to-cell contacts and is thus similar to the environment of mature chondrocytes in cartilage [37]. ...
By directly monitoring single cell growth in a microfluidic platform, we interrogated genome-deletion effects in Escherichia coli strains. We compared the growth dynamics of a wild type strain with a clean genome strain, and their derived mutants at the single-cell level. A decreased average growth rate and extended average lag time were found for the clean genome strain, compared to those of the wild type strain. Direct correlation between the growth rate and lag time of individual cells showed that the clean genome population was more heterogeneous. Cell culturability (the ratio of growing cells to the sum of growing and non-growing cells) of the clean genome population was also lower. After random mutations induced by a glucose starvation treatment in chemostat culture, the average growth rate and cell culturability increased, and the lag time and heterogeneity decreased. However, the wild type mutants retained a high diversity of growth phenotype. These results suggest that quasi-essential genes that were deleted in the clean genome might be required to retain a diversity of growth characteristics at the individual cell level under environmental stress. These observations highlight that single-cell microfluidics can reveal subtle individual cellular responses, enabling in-depth understanding of the population.
... In addition, the complex hierarchical structure involving both articular cartilage and underlying subchondral bone is another huge challenge in reconstruction of osteochondral tissue. Several techniques are currently used to treat osteochondral defects, including autografts transplantation [4,5], autologous chondrocytes transplantation [6,7] and marrow stimulation such as subchondral drilling [8] and microfracture [9]. Although successful in some aspects, each of these techniques has its own limitations. ...
Osteochondral defects cannot be adequately self-repaired due to the presence of the sophisticated hierarchical structure and the lack of blood supply in cartilage. Thus, one of the major challenges remaining in this field is the structural design of a biomimetic scaffold that satisfies the specific requirements for osteochondral repair. To address this hurdle, a bio-inspired multilayer osteochondral scaffold that consisted of the poly(ε-caprolactone) (PCL) and the hydroxyapatite (HA)/PCL microspheres, was constructed via selective laser sintering (SLS) technique. The SLS-derived scaffolds exhibited an excellent biocompatibility to support cell adhesion and proliferation in vitro. The repair effect was evaluated by implanting the acellular multilayer scaffolds into osteochondral defects of a rabbit model. Our findings demonstrated that the multilayer scaffolds were able to induce articular cartilage formation by accelerating the early subchondral bone regeneration, and the newly formed tissues could well integrate with the native tissues. Consequently, the current study not only achieves osteochondral repair, but also suggests a promising strategy for the fabrication of bio-inspired multilayer scaffolds with well-designed architecture and gradient composition via SLS technique.
... This flexible cytoskeleton appears to be critical in maintaining constant cell volume during the 21 day culture, and encouraged redifferentiation. This phenomenon highlights the critical influences on cell fate due to the variations in seeding density and cell-matrix holding time [13,45], since they all affect initial cell morphology in a matrix. Compared to the majority of 3D culture systems, the current single cell 3D culture eliminates cell-to-cell contacts and is thus similar to the environment of mature chondrocytes in cartilage [37] . ...
Dedifferentiation of chondrocytes during in vitro expansion remains an unsolved challenge for repairing serious articular cartilage defects. In this study, a novel culture system was developed to modulate single cell geometry in 3D and investigate its effects on the chondrocyte phenotype. The approach uses 2D micropatterns followed by in situ hydrogel formation to constrain single cell shape and spreading. This enables independent control of cell geometry and extracellular matrix. Using collagen I matrix, we demonstrated the formation of a biomimetic collagenous "basket" enveloping individual chondrocytes cells. By quantitatively monitoring the production by single cells of chondrogenic matrix (e.g. collagen II and aggrecan) during 21-day cultures, we found that if the cell's volume decreases, then so does its cell resistance to dedifferentiation (even if the cells remain spherical). Conversely, if the volume of spherical cells remains constant (after an initial decrease), then not only do the cells retain their differentiated status, but previously de-differentiated redifferentiate and regain a chondrocyte phenotype. The approach described here can be readily applied to pluripotent cells, offering a versatile platform in the search for niches toward either self-renewal or targeted differentiation.
Copyright © 2015. Published by Elsevier Ltd.
... Chondrocyte expansion in monolayer culture enables rapid cell harvesting, and is commonly used in classic autologous chondrocyte transplantation. However, dedifferentiation occurs as early as in the first passage, becoming dominant after a few serial passages [4,5] . Currently, this problem has been addressed by two approaches. ...
Autologous chondrocyte transplantation (ACT) has become a promising method for repairing large articular defects. However, dedifferentiation of chondrocytes during cell expansion remains a major limitation for ACT procedures. In this study, we explore the potential of confining cell shape for re-differentiation of dedifferentiated bovine chondrocytes. A novel culture system, combining 2D micropatterning with 3D matrix formation, was developed to control and maintain individual chondrocyte's shape. Both collagen II synthesis and the mechanical properties of cells were monitored during re-differentiation. We show that a spherical morphology without cell spreading plays a limited role in induction of re-differentiation. Instead, isolated, dedifferentiated chondrocytes partially regain chondrogenic properties if they have an appropriate cell shape and limited spreading.
... Extended cell cultures typically result in a significant loss of the chondrogenic phenotype in vitro and the ability to form hyaline cartilage in vivo. 10,30 The problem of dedifferentiating chondrocytes is further reflected in the fact that although clinical products for autologous chondrocyte transplantation are designed with an upper limit to the level of monolayer expansion (the exact process parameters are not publicly available), the resulting constructs still exhibit signs of dedifferentiation. 31 As current gene expression analysis indicated, robust redifferentiation of culture expanded ACs remains an elusive goal, and it should therefore be beneficial to limit the degree of dedifferentiation in the first place. ...
In this work, we evaluated the ability of 3D co-cultures with mesenchymal stem cells (MSCs) to redifferentiate monolayer expanded articular chondrocytes (ACs) and produce cartilaginous extracellular matrix at varying stages of the dedifferentiation process and further examined the dependency of this effect on the culture medium composition. Primary bovine ACs were expanded in monolayers for up to nine population doublings to obtain seven cell stocks with gradually increasing levels of dedifferentiation. Culture expanded ACs were then seeded as monocultures and co-cultures with rabbit bone marrow-derived MSCs (30:70 ratio of ACs-to-MSCs) on porous scaffolds. Parallel cultures were established for each cell population in serum-containing growth medium and serum-free induction medium supplemented with dexamethasone and TGF-β3. After 3 weeks, all groups were analyzed for DNA content, glycosaminoglycan (GAG) and hydroxyproline (HYP) production, and chondrogenic gene expression. Significant enhancements in cellularity, GAG content and GAG/HYP ratio, and chondrogenic phenotype were observed in the induction medium compared to growth medium at all levels of AC expansion. Furthermore, primary co-cultures showed similarly enhanced chondrogenesis compared to monocultures in both culture media, whereas passaged ACs benefitted from co-culturing only in the induction medium. We conclude that co-cultures of ACs and MSCs can produce superior in vitro engineered cartilage in comparison to pure AC cultures, due to both heterotypic cellular interactions and decreased need for monolayer expansion of biopsied chondrocytes. While the initial level of AC dedifferentiation affected the quality of the engineered constructs, co-culture benefits were realized at all stages of AC expansion when suitable chondroinductive culture medium was used.
... Much higher DNA and matrix contents were observed with primary chondrocytes than with passage two cells, consistent with the notion of rapid dedifferentiation of culture expanded chondrocytes [25][26][27]. In addition, passaged ACs showed decreasing collagen expression and unchanged GAG contents from two to four weeks, further highlighting the problem of dedifferentiation during prolonged cultures. ...
In this work, articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) with 1:1 and 1:3 cell ratios were co-cultured in order to evaluate if a majority of primary ACs can be replaced with MSCs without detrimental effects on in vitro chondrogenesis. We further used a xenogeneic culture model to study if such co-cultures can result in redifferentiation of passaged ACs. Cells were cultured in porous scaffolds for four weeks and their cellularity, cartilage-like matrix formation and chondrogenic gene expression levels (collagen I and II, aggrecan) were measured. Constructs with primary bovine ACs had ~1.6 and 5.5 times higher final DNA and glycosaminoglycan contents, respectively, in comparison to those with culture expanded chondrocytes or MSCs harvested from the same animals. Equally robust chondrogenesis was also observed in co-cultures, even when up to 75% of primary ACs were initially replaced with MSCs. Furthermore, species-specific RT-PCR analysis indicated a gradual loss of MSCs in bovine-rabbit co-cultures. Finally, co-cultures using primary and culture expanded ACs resulted in similar outcomes. We conclude that the most promising cell source for cartilage engineering was the co-cultures, as the trophic effect of MSCs may highly increase the chondrogenic potential of ACs thus diminishing the problems with primary chondrocyte harvest and expansion.
... The possibility forFig. 3 Collagen type II and aggrecan expression were compared in the groups B20 years and [20 years, showing a statistically significant difference with 55 and 56 % increase in the cartilage markers, respectively (p \ 0.02) Knee Surg Sports Traumatol Arthrosc extrapolation of marker expression in different passages and cultures of chondrocytes has been previously shown in rabbits [26]. Depending on the statistical model used, the border was determined in a range between 18 and 20 years. ...
Purpose
Autologous chondrocyte implantation (ACI) is a well-established treatment method for cartilage defects in knees. Age-related grouping was based on expression data of cartilage-specific markers. Specificities of ACI in the different populations were analysed.
Methods
Two hundred and sixty-seven patients undergoing ACI in the knee between 2006 and 2010 were included in this analysis. Cell characteristics and expression data of cartilage-specific surface markers as CD44, aggrecan and collagen type II were statistically analysed for age association. Epidemiological data of the defined groups were compared. Course of treatment was evaluated using MRI.
Results
A correlation analysis showed statistically significant associations between age and aggrecan or collagen type II expression in all patients <30 years. A cluster analysis could predict age-dependent expression of these markers separating groups with an average age of 18.1 ± 2.3 and 23.6 ± 4.2 years, respectively (p < 0.02). Discriminance analysis suggested the age border between adults and juveniles at about 20 years. There was no influence of age on cell characteristics or CD44 expression. In the 19 of 267 patients with an age ≤18 years, gender distribution was not different compared to adults, but patella was significantly more affected. Cartilage lesions were mainly caused by osteochondritis dissecans (OCD) and trauma. The Knee Osteoarthritis Scoring System in MRI reached 4.8 ± 2.3 points before, declining to 3.3 ± 2.3 points 6 and 12 months after the operation.
Conclusions
Age-related expression of cartilage-specific markers allows definition of adolescents in cartilage regenerating surgery. Chondromalacia in these patients is mainly caused by OCD or trauma.
Level of evidence
Case series, Level IV.
Tissue engineering provides a new approach for the treatment of osteochondral defects. However, the lack of an ideal double-layer scaffold with osteochondral-biomimetic microenvironment and interface similar to native articular tissue greatly limits clinical translation. Our current study developed a double-layer acellular osteochondral matrix (AOM) scaffold with natural osteochondral-biomimetic microenvironment and interface by integrating ultraviolet (UV) laser and decellularization techniques. The laser parameters were optimized to achieve a proper pore depth close to the osteochondral interface, which guaranteed complete decellularization, sufficient space for cell loading, and relative independence of the chondrogenic and osteogenic microenvironments. Gelatin-methacryloyl (GelMA) hydrogel was further used as the cell carrier to significantly enhance the efficiency and homogeneity of cell loading in the AOM scaffold with large pore structure. Additionally, in vitro results demonstrated that the components of the AOM scaffold could efficiently regulate the chondrogenic/osteogenic differentiations of bone marrow stromal cells (BMSCs) by activating the chondrogenic/osteogenic related pathways. Importantly, the AOM scaffolds combined with BMSC-laden GelMA hydrogel successfully realized tissue-specific repair of the osteochondral defects in a knee joint model of rabbit. The current study developed a novel double-layer osteochondral biomimetic scaffold and feasible strategy, providing strong support for the tissue-specific repair of osteochondral defects and its future clinical translation.
The objective of an articular cartilage repair treatment is to repair the affected surface of an articular joint’s hyaline cartilage. Currently, both biological and tissue engineering research is concerned with discovering the clues needed to stimulate cells to regenerate tissues and organs totally or partially. The latest findings on nanotechnology advances along with the processability of synthetic biomaterials have succeeded in creating a new range of materials to develop into the desired biological responses to the cellular level. 3D printing has a great ability to establish functional tissues or organs to cure or replace abnormal and necrotic tissue, providing a promising solution for serious tissue/organ failure. The 4D print process has the potential to continually revolutionize the current tissue and organ manufacturing platforms. A new active research area is the development of intelligent materials with high biocompatibility to suit 4D printing technology. As various researchers and tissue engineers have demonstrated, the role of growth factors in tissue engineering for repairing osteochondral and cartilage defects is a very important one. Following animal testing, cell-assisted and growth-factor scaffolds produced much better results, while growth-free scaffolds showed a much lower rate of healing.
DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full -scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome-wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5-azacytidine (5-AzaC). This research revealed that the general methylation level of CpG was increased while the COL-1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5-AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL-1A1 promoter was increased after 5-AzaC treatment. The COL-1A1 expression level was increased while that of SOX-9 was decreased during the chondrocyte dedifferentiation. 5-AzaC treatment up-regulated the SOX-9 expression while down-regulated the COL-1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage-specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. This article is protected by copyright. All rights reserved.
Articular cartilage defects are among the most common disabling conditions of humans in the western world. The most prominent risk factors are age, weight, and trauma. Osteoarthritis is the typical outcome of chronic articular cartilage defects and up to date cartilage regeneration remains elusive. The discovery of amniotic fluid-derived stem cells has opened a multitude of new therapeutic options, one of them being their use for novel stem cell-based tissue regeneration techniques in order to treat cartilage defects. Here, we summarize developmental stages and growth factors as well as extracellular molecules involved in articular cartilage formation as well as degeneration. Furthermore, we discuss recent advances in the use of amniotic fluid stem cells as novel cell sources for the generation of cartilage tissue and how the endogenous cartilage formation process could be recapitulated during artificial tissue engineering. © 2014 Springer Science+Business Media New York. All rights reserved.
We aimed to build a culture model of chondrocytes in vitro, and to study the differential properties between fibrochondrocytes and hyaline chondrocytes. Histological sections were stained with haematoxylin and eosin so that we could analyse the histological structure of the fibrocartilage and hyaline cartilage. Condylar fibrochondrocytes and femoral hyaline chondrocytes were cultured from four, 4-week-old, New Zealand white rabbits. The production of COL2A1, COL1OA1, SOX9 and aggrecan was detected by real time-q polymerase chain reaction (RT-qPCR) and immunoblotting and the differences between them were compared statistically. Histological structures obviously differed between fibrocartilage and hyaline cartilage. COL2A1 and SOX9 were highly expressed within cell passage 2 (P2) of both fibrochondrocytes and hyaline chondrocytes, and reduced significantly after cell passage 4 (P4). The mRNA expressions of COL2A1 (p=0.05), COL10A1 (p=0.04), SOX9 (p=0.03), and aggrecan (p=0.04) were significantly higher in hyaline chondrocytes than in fibrochondrocytes, whereas the expression of COL1A1 (p=0.02) was the opposite. Immunoblotting showed similar results. We have built a simple and effective culture model of chondrocytes in vitro, and the P2 of chondrocytes is recommended for further studies. Condylar fibrocartilage and femoral hyaline cartilage have unique biological properties, and the regulatory mechanisms of endochondral ossification for the condyle should be studied independently in the future.
Copyright © 2014 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Aim:
We aimed to investigate freshly isolated compared with culture-expanded chondrocytes with respect to early regenerative response, cytokine production and cartilage formation in response to four commonly used biomaterials.
Materials & methods:
Chondrocytes were both directly and after expansion to passage 2, incorporated into four biomaterials: Polyactive™, Beriplast®, HyStem® and a type II collagen gel. Early cartilage matrix gene expression, cytokine production and glycosaminoglycan (GAG) and DNA content in response to these biomaterials were evaluated.
Results:
HyStem induced more GAG production, compared with all other biomaterials (p ≤ 0.001). Nonexpanded cells did not always produce more GAGs than expanded chondrocytes, as this was biomaterial-dependent. Cytokine production and early gene expression were not predictive for final regeneration.
Conclusion:
For chondrocyte-based cartilage treatments, the biomaterial best supporting cartilage matrix production will depend on the chondrocyte differentiation state and cannot be predicted from early gene expression or cytokine profile.
Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration (1). Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery (2-4). Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies. The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) (5). Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation (6). In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular (7-9). MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration (10). The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics (11). The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.
Articular cartilage regeneration poses particularly tough challenges for implementing cell-based therapies. Many cell types have been investigated looking for a balanced combination of responsiveness and stability, yet techniques are still far from defining a gold standard. The work presented focuses on the reliable expansion and characterization of a clinical grade human epiphyseal chondroprogenitor (ECP) cell bank from a single tissue donation. A parental human ECP cell bank was established, which provides the seed material for master and working cell banks. ECPs were investigated at both low and high cumulative population doublings looking at morphology, monolayer expansion kinetics, resistance to cryogenic shock, colony-forming efficiency, and cell surface markers. Three-dimensional micropellet assays were used to determine spontaneous extracellular matrix deposition at varying population doublings and monolayer 2D differentiation studies were undertaken to assess the propensity for commitment into other lineages and their stability. ECPs exhibited remarkable homogeneity in expansion with a steady proliferative potential averaging three population doublings over 8 days. Surface marker analysis revealed no detectable contaminating subpopulations or population enrichment during prolonged culture periods. Despite a slight reduction in Sox9 expression levels at higher population doublings in monolayer, nuclear localization was equivalent both in monolayer and in micropellet format. Equally, ECPs were capable of depositing glycosaminoglycans and producing aggrecan, collagen I, and collagen II in 3D pellets both at low and high population doublings indicating a stable spontaneous chondrogenic potential. Osteogenic induction was differentially restricted in low and high population doublings as observed by Von Kossa staining of calcified matrix, with a notable collagen X, MMP13, and ADAMTS5 downregulation. Rare adipogenic induction was seen as evidenced by cytoplasmic lipid accumulation detectable by Oil Red O staining. These findings highlight the reliability, stability, and responsiveness of ECPs over prolonged culture, making them ideal candidates in defining novel strategies for cartilage regeneration.
Chronic articular cartilage defects are the most common disabling conditions of humans in the western world. The incidence for cartilage defects is increasing with age and the most prominent risk factors are overweight and sports associated overloading. Damage of articular cartilage frequently leads to osteoarthritis due to the aneural and avascular nature of articular cartilage, which impairs regeneration and repair. Hence, patients affected by cartilage defects will benefit from a cell-based transplantation strategy. Autologous chondrocytes, mesenchymal stem cells and embryonic stem cells are suitable donor cells for regeneration approaches and most recently the discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options. It is the aim of this review to summarize recent advances in the use of amniotic fluid stem cells as novel cell sources for the treatment of articular cartilage defects. Molecular aspects of articular cartilage formation as well as degeneration are summarized and the role of growth factor triggered signaling pathways, scaffolds, hypoxia and autophagy during the process of chondrogenic differentiation are discussed.
Summary Objective: Data pertaining to the quantitative structural features and organization of normal articular cartilage are of great importance in understanding its biomechanical properties and in attempting to establish this tissue's counterpart by engineering in vitro. A comprehensive set of such baseline data is, however, not available for humans. It was the purpose of the present study to furnish the necessary information. Design: The articular cartilage layer covering the medial femoral condyle of deceased persons aged between 23 and 49 years was chosen for the morphometric analysis of cell parameters using confocal microscopy in conjunction with unbiased stereological methods. The height of the hyaline articular cartilage layer, as well as that of the calcified cartilage layer and the subchondral bone plate, were also measured. Results: The mean height of the hyaline articular cartilage layer was found to be 2.4 mm, the volume density of chondrocytes therein being 1.65%, the number of cells per mm3 of tissue 9626 and the mean cell diameter 13 m. Other estimators (including matrix mass per cell and cell profile density) were also determined. Conclusions: A comparison of these normal human quantitative data with those published for experimental animals commonly used in orthopaedic research reveals substantial differences, consideration of which in tissue engineering strategies destined for human application are of paramount importance for successful repair. © 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.
Autologous chondrocyte implantation (ACI) is a well-established therapeutic option for the treatment of cartilage defects of the knee joint. Since information concerning the cellular aspects of ACI is still limited, the aim of the present study was to investigate relevant differences between chondrocyte quality after in vitro cultivation and possible correlations with patient-specific factors.
Cell quality of 252 consecutive ACI patients was assessed after chondrocyte in vitro expansion by determination of the expression of cartilage relevant surface marker CD44 and cartilage-specific differentiation markers (aggrecan and collagen type II). All cell quality parameters were correlated with patient-specific parameters, such as age, size and defect location, number of defects and grade of joint degeneration according to the Kellgren-Lawrence classification.
Neither the expression of CD44, aggrecan or collagen type II nor cell density or viability after proliferation seemed to correlate with the grade of joint degeneration, defect aetiology or patient gender. However, chondrocytes harvested from the knee joints of patients at less than 20 years of age showed significantly higher expression rates of cartilage-specific markers when compared to older patients' chondrocytes.
The present study identifies relevant differences concerning chondrocyte quality after in vitro expansion in a highly preselected study population of 252 patients that from a surgical point of view were eligible for ACI. With the exception of patients aged 20 years or younger, no patient-specific parameters could be identified which might allow anticipation of cell quality in individual patients.
Although there is much known about the role of BMPs in cartilage metabolism reliable data about the in vivo regulation in natural and surgically induced cartilage repair are still missing.
Lavage fluids of knee joints of 47 patients were collected during surgical therapy. 5 patients had no cartilage lesion and served as a control group, the other 42 patients with circumscribed cartilage defects were treated by microfracturing (19) or by an Autologous Chondrocyte Implantation (23). The concentrations of BMP-2 and BMP-7 were determined by ELISA. The clinical status was evaluated using the IKDC Score prior to and 1 year following the operation.
High level expression in the control group was found for BMP-2, concentrations of BMP-7 remained below detection levels. No statistical differences could be detected in concentrations of BMP-2 or BMP-7 in the lavage fluids of knees with cartilage lesions compared to the control group. Levels of BMP-7 did not change after surgical cartilage repair, whereas concentrations of BMP-2 statistically significant increased after the intervention (p < 0.001). The clinical outcome following cartilage regenerating surgery increased after 1 year by 29% (p < 0.001). The difference of the IKDC score after 1 year and prior to the operation was used to quantify the degree of improvement following surgery. This difference statistically significant correlated with initial BMP-2 (R = 0.554, p < 0.001) but not BMP-7 (R = 0.031, n.s.) levels in the knee joints.
BMP-2 seems to play an important role in surgically induced cartilage repair; synovial expression correlates with the clinical outcome.
The medium-term results of autologous chondrocyte implantation (ACI) have shown good to excellent outcomes for the majority of patients. However, no long-term results 10 to 20 years after the surgery have been reported.
Autologous chondrocyte implantation provides a durable solution to the treatment of full-thickness cartilage lesions of the knee, maintaining good clinical results even 10 to 20 years after implantation.
Case series; Level of evidence, 4.
In this uncontrolled study, questionnaires with the Lysholm, Tegner-Wallgren, Brittberg-Peterson, modified Cincinnati (Noyes), and Knee Injury and Osteoarthritis Outcome Score (KOOS) scores were sent to 341 patients. Preoperative Lysholm, Tegner-Wallgren, and Brittberg-Peterson scores were also retrieved when possible from patients' files. The patients were asked to grade their status during the past 10 years as better, worse, or unchanged. Finally, they were asked if they would do the operation again.
There were 224 of 341 patients who replied to our posted questionnaires and were assessed. The mean cartilage lesion size was 5.3 cm(2). Ten to 20 years after the implantation (mean, 12.8 years), 74% of the patients reported their status as better or the same as the previous years. There were 92% who were satisfied and would have the ACI again. The Lysholm, Tegner-Wallgren, and Brittberg-Peterson scores were improved compared with the preoperative values. The average Lysholm score improved from 60.3 preoperatively to 69.5 postoperatively, the Tegner from 7.2 to 8.2, and the Brittberg-Peterson from 59.4 to 40.9. At the final measurement, the KOOS score was on average 74.8 for pain, 63 for symptoms, 81 for activities of daily living (ADL), 41.5 for sports, and 49.3 for quality of life (QOL). The average Noyes score was 5.4. Patients with bipolar lesions had a worse final outcome than patients with multiple unipolar lesions. The presence of meniscal injuries before ACI or history of bone marrow procedures before the implantation did not appear to affect the final outcomes. The age at the time of the operation or the size of lesion did not seem to correlate with the final outcome.
Autologous chondrocyte implantation has emerged as an effective and durable solution for the treatment of large full-thickness cartilage and osteochondral lesions of the knee joint. Our study suggests that the clinical and functional outcomes remain high even 10 to 20 years after the implantation.
Since the first patient was implanted with autologous cultured chondrocytes more than 20 years ago, new variations of cell therapies for cartilage repair have appeared. Autologous chondrocyte implantation, a first-generation cell therapy, uses suspended autologous cultured chondrocytes in combination with a periosteal patch. Collagen-covered autologous cultured chondrocyte implantation, a second-generation cell therapy, uses suspended cultured chondrocytes with a collagen type I/III membrane. Today's demand for transarthroscopic procedures has resulted in the development of third-generation cell therapies that deliver autologous cultured chondrocytes using cell carriers or cell-seeded scaffolds.
To review the current evidence of the matrix-induced autologous chondrocyte implantation procedure, the most widely used carrier system to date. Also discussed are the characteristics of type I/III collagen membranes, behavior of cells associated with the membrane, surgical technique, rehabilitation, clinical outcomes, and quality of repair tissue.
Systematic review.
Relevant publications were identified by searching Medline from its inception (1949) to December 2007; peer-reviewed publications of preclinical and clinical cell behavior, manufacturing process, surgical technique, and rehabilitation protocols were identified. Preclinical and clinical studies were included if they contained primary data and used a type I/III collagen membrane.
Data from these studies demonstrate that patients treated with matrix-induced autologous chondrocyte implantation have an overall improvement in clinical outcomes. Reduced visual analog scale pain levels (range, 1.7-5.32 points) and improvements in the modified Cincinnati (range, 3.8-34.2 points), Lysholm-Gillquist (range, 23.09-47.6 points), Tegner-Lysholm (range, 1.39-3.9 points), and International Knee Documentation Classification scale (P <.05) were observed. Patients had good-quality (hyaline-like) repair tissue as assessed by arthroscopic evaluation (including International Cartilage Repair Society score), magnetic resonance imaging, and histology, as well as a low incidence of postoperative complications.
The findings suggest that matrix-induced autologous chondrocyte implantation is a promising third-generation cell therapy for the repair of symptomatic, full-thickness articular cartilage defects.
Autologous chondrocyte implantation (ACI) is frequently used to treat symptomatic defects of the articular cartilage.
To test whether matrix-associated autologous chondrocyte implantation or the original periosteal flap technique provides superior outcomes in terms of clinical efficacy and safety.
Randomized controlled trial; Level of evidence, 2.
Twenty-one patients (mean age, 29.3 +/- 9.1 years) with symptomatic isolated full-thickness cartilage defects (mean 4.1 +/- 09 cm2) at the femoral condyle were randomized to matrix-associated autologous chondrocyte implantation or the original periosteal flap technique. The primary outcome parameter was the postoperative change in knee function as assessed by the International Knee Documentation Committee (IKDC) score at 12 months after ACI. In addition, the IKDC score was assessed at 3, 6, 12, and 24 months after surgery. Secondary outcome parameters were postoperative changes in health related quality of life (Short Form-36 Health Survey), knee functionality (Lysholm and Gillquist score), and physical activity (Tegner Activity Score) at 3, 6, 12, and 24 months after ACI. Magnetic resonance imaging was performed to evaluate the cartilage 6, 12, and 24 months after ACI and rated using the Magnetic Resonance Observation of Cartilage Repair Tissue score. Adverse events were recorded to assess safety.
The primary outcome parameter showed improvement of patients 1 year after autologous chondrocyte implantation, but there was no difference between the periosteal flap technique and matrix-associated ACI (P = .5573); 2 years after ACI, a similar result was found (P = .4994). The study groups did not show differences in the Short Form-36 categories and in knee functionality as assessed by Tegner Activity Score 12 months (P = .4063) and 24 months (P = .1043) after ACI. There was a significant difference in the Lysholm and Gillquist score at 12 months (P = .0449) and 24 months (P = .0487) favoring the periosteal flap technique group. At 6 months after surgery, a significantly lower Magnetic Resonance Observation of Cartilage Repair score was obtained in the matrix-associated ACI group (P = .0123), corresponding to more normal magnetic resonance imaging diagnostic findings. Twelve and 24 months after ACI, the differences between the 2 groups were not significant (12 months, P = .2065; 24 months, P = .6926). Adverse events were related to knee problems such as transplant delamination, development of an osseous spur, osteochondral dissection, and transplant hypertrophy. Systemic (allergic, toxic, or autoimmune) reactions did not occur.
There was no difference in the efficacy between the original and the advanced ACI technique 12 and 24 months after surgery regarding International Knee Documentation Committee, Tegner Activity Score, and Short Form-36; however, with respect to the Lysholm and Gillquist score, better efficacy was observed in the periosteal flap technique group.
Damaged articular cartilage has limited capacity for self-repair. Autologous chondrocyte implantation using a characterized cell therapy product results in significantly better early structural repair as compared with microfracture in patients with symptomatic joint surface defects of the femoral condyles of the knee.
Purpose: To evaluate. clinical outcome at 36 months after characterized chondrocyte implantation (CCI) versus microfracture (MF).
Study Design: Randomized controlled trial; Level of evidence, 1.
Methods: Patients aged 18 to 50 years with single International Cartilage Repair Society (ICRS) grade III/IV symptomatic cartilage defects of the femoral condyles were randomized to CCI (n = 57) or MF (n = 61). Clinical outcome was measured over 36 months by the Knee injury and Osteoarthritis Outcome Score (KOOS). Serial magnetic resonance imaging (MRI) scans were scored using the Magnetic resonance Observation of Cartilage Repair Tissue (MOCART) system and 9 additional items. Gene expression profile scores associated with ectopic cartilage formation were determined by RT-PCR.
Results: Baseline mean overall KOOS (+/- SE) was comparable between the CCI and MF groups (56.30 +/- 1.91 vs 59.46 +/- 1.98, respectively). Mean improvement (+/- SE) from baseline to 36 months in overall KOOS was greater in the CCI group than the MF group (21.25 +/- 3.60 vs 15.83 +/- 3.48, respectively), while in a mixed linear model analysis with time as a categorical variable, significant differences favoring CCI were shown in overall KOOS (P = .048) and the subdomains of Pain (P = .044) and QoL (P = .036). More CCI- than MF-treated patients were treatment responders (83% vs 62%, respectively). In patients with symptom onset of
Mechanical and chemical stimulation have been shown to enhance in vitro chondrogenesis. The aim of this study was to analyze and compare combined physicobiochemical effects. Bovine articular chondrocytes were retrovirally transduced to express bone morphogenetic protein-2 (BMP-2) or left as naïve controls. Cells were seeded in three-dimensional polyurethane scaffolds and further cultured under static conditions or exposed to dynamic compression and shear in a joint-specific bioreactor. Four groups: control (A), load (B), BMP-2-infected (C), and BMP-2-infected plus load (D) were analyzed for DNA and glycosaminoglycan (GAG) content; collagen I, II, and X; aggrecan, (cartilage oligomeric protein (COMP), superficial zone protein, matrix metalloproteinase (MMP)-3; MMP-13 mRNA; histology; and immunohistochemistry at 7, 21, and 35 days post-seeding. Synergistic effects (D) were higher than the sum of the individual treatments (B and C) for GAG/DNA, collagen II, and COMP. Histology revealed a functional organization in D including an intense safranin O staining in C and D superior to that in A and B. Immunostaining for collagen II and aggrecan was detected in C and D and was strongest in D. The results show that both stimuli augment in vitro chondrogenesis better than in controls. Biochemical manipulation proved to be predominantly more effective than load, and synergistic effects were demonstrated.
The present study established characteristics of tissue regrowth in patients suffering knee lesions treated with grafts of autologous chondrocytes grown on three-dimensional hyaluronic acid biomaterials.
This multicentred study involved a second-look arthroscopy/biopsy, 5 to 33 months post implant (n = 63). Seven patients allowed a third-look biopsy, three of which were performed 18 months post implant. Characteristics of tissues were histologically and histochemically evaluated. The remaining bone stubs were evaluated for cartilage/bone integration. For data analysis, biopsies were further divided into those obtained from postoperative symptomatic patients (n = 41) or from asymptomatic patients (n = 22).
The percentage of hyaline regenerated tissues was significantly greater in biopsies obtained after, versus within, 18 months of implantation. Differences were also observed between symptomatic and asymptomatic patients: reparative tissues taken from symptomatic patients 18 months after grafting were mainly fibrocartilage or mixed (hyaline-fibrocartilage) tissue, while tissues taken from asymptomatic patients were hyaline cartilage in 83% of biopsies. In a small group of asymptomatic patients (n = 3), second-look and third-look biopsies taken 18 months after surgery confirmed maturation of the newly formed tissue over time. Cartilage maturation occurred from the inner regions of the graft, in contact with subchondral bone, towards the periphery of the implant.
The study indicates that, in asymptomatic patients after chondrocyte implantation, regenerated tissue undergoes a process of maturation that in the majority of cases takes longer than 18 months for completion and leads to hyaline tissue and not fibrous cartilage. Persistence of symptoms might reflect the presence of a nonhyaline cartilage repair tissue.
Full-thickness defects of articular cartilage in the knee have a poor capacity for repair. They may progress to osteoarthritis and require total knee replacement. We performed autologous chondrocyte transplantation in 23 people with deep cartilage defects in the knee.
The patients ranged in age from 14 to 48 years and had full-thickness cartilage defects that ranged in size from 1.6 to 6.5 cm2. Healthy chondrocytes obtained from an uninvolved area of the injured knee during arthroscopy were isolated and cultured in the laboratory for 14 to 21 days. The cultured chondrocytes were then injected into the area of the defect. The defect was covered with a sutured periosteal flap taken from the proximal medial tibia. Evaluation included clinical examination according to explicit criteria and arthroscopic examination with a biopsy of the transplantation site.
Patients were followed for 16 to 66 months (mean, 39). Initially, the transplants eliminated knee locking and reduced pain and swelling in all patients. After three months, arthroscopy showed that the transplants were level with the surrounding tissue and spongy when probed, with visible borders. A second arthroscopic examination showed that in many instances the transplants had the same macroscopic appearance as they had earlier but were firmer when probed and similar in appearance to the surrounding cartilage. Two years after transplantation, 14 of the 16 patients with femoral condylar transplants had good-to-excellent results. Two patients required a second operation because of severe central wear in the transplants, with locking and pain. A mean of 36 months after transplantation, the results were excellent or good in two of the seven patients with patellar transplants, fair in three, and poor in two; two patients required a second operation because of severe chondromalacia. Biopsies showed that 11 of the 15 femoral transplants and 1 of the 7 patellar transplants had the appearance of hyaline cartilage.
Cultured autologous chondrocytes can be used to repair deep cartilage defects in the femorotibial articular surface of the knee joint.
Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the tissue-engineered cartilage in existence has been successful in mimicking the morphological and biochemical appearance of hyaline cartilage, it is generally mechanically inferior to the natural tissue. In this study, we tested the hypothesis that the application of dynamic deformational loading at physiological strain levels enhances chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more functional engineered tissue construct than in free swelling controls. A custom-designed bioreactor was used to load cell-seeded agarose disks dynamically in unconfined compression with a peak-to-peak compressive strain amplitude of 10 percent, at a frequency of 1 Hz, 3 x (1 hour on, 1 hour off)/day, 5 days/week for 4 weeks. Results demonstrated that dynamically loaded disks yielded a sixfold increase in the equilibrium aggregate modulus over free swelling controls after 28 days of loading (100 +/- 16 kPa versus 15 +/- 8 kPa, p < 0.0001). This represented a 21-fold increase over the equilibrium modulus of day 0 (4.8 +/- 2.3 kPa). Sulfated glycosaminoglycan content and hydroxyproline content was also found to be greater in dynamically loaded disks compared to free swelling controls at day 21 (p < 0.0001 and p = 0.002, respectively).
One tissue-engineering approach being investigated for the treatment of defects in articular cartilage involves the implantation of autologous chondrocyte-seeded absorbable scaffolds. The present study evaluated the effects of passage number (freshly isolated and passages 1 and 2) and collagen type on the proliferative, biosynthetic, and contractile activity of adult canine articular chondrocytes grown in type I and II collagen-glycosaminoglycan (GAG) matrices that were cross-linked by dehydrothermal/carbodiimide treatment. P0, P1, and P2 cells seeded in the type II matrices continued to proliferate over a 4-week period, but thereafter the P0 and P1 cells continued to increase in number and the P2 cells decreased. At 4 weeks the DNA contents of the type I and II matrices seeded with P1 and P2 cells were comparable, and higher than the values for matrices seeded with freshly isolated chondrocytes. The rates of protein and GAG synthesis by the P1 and P2 cells were comparable, and higher than the rates for the P0 chondrocytes, after 1 week, and the rates were generally higher in the type II than in the type I collagen scaffolds. Western blot analysis demonstrated the presence of newly synthesized type II collagen in type II matrices in which P1 and P2 cells were grown. The cross-linking treatment imparted a sufficient degree of mechanical stiffness to both types of matrices to resist cell-mediated contraction. This study demonstrated that adult articular chondrocytes expanded in number through two passages in monolayer culture can be expected to provide behavior comparable to or better than freshly isolated cells with respect to proliferation and biosynthesis through 4 weeks of culture in collagen-GAG matrices, and these cells retain the capability to synthesize type II collagen. The results of this investigation further commend the use of a type II collagen-GAG matrix, based on the higher biosynthetic rates of the cells grown in the matrices, for the preparation of chondrocyte-seeded scaffolds for articular cartilage tissue engineering.
Autologous chondrocyte implantation (ACI) is used widely as a treatment for symptomatic chondral and osteochondral defects of the knee. Variations of the original periosteum-cover technique include the use of porcine-derived type I/type III collagen as a cover (ACI-C) and matrix-induced autologous chondrocyte implantation (MACI) using a collagen bilayer seeded with chondrocytes. We have performed a prospective, randomised comparison of ACI-C and MACI for the treatment of symptomatic chondral defects of the knee in 91 patients, of whom 44 received ACI-C and 47 MACI grafts.
Both treatments resulted in improvement of the clinical score after one year. The mean modified Cincinnati knee score increased by 17.6 in the ACI-C group and 19.6 in the MACI group (p = 0.32). Arthroscopic assessments performed after one year showed a good to excellent International Cartilage Repair Society score in 79.2% of ACI-C and 66.6% of MACI grafts. Hyaline-like cartilage or hyaline-like cartilage with fibrocartilage was found in the biopsies of 43.9% of the ACI-C and 36.4% of the MACI grafts after one year. The rate of hypertrophy of the graft was 9% (4 of 44) in the ACI-C group and 6% (3 of 47) in the MACI group. The frequency of re-operation was 9% in each group.
We conclude that the clinical, arthroscopic and histological outcomes are comparable for both ACI-C and MACI. While MACI is technically attractive, further long-term studies are required before the technique is widely adopted.
We investigated whether, and under which conditions (i.e., cell-seeding density, medium supplements), in vitro preculture enhances in vivo development of human engineered cartilage in an ectopic nude mouse model. Monolayer-expanded adult human articular chondrocytes (AHACs) were seeded into Hyalograft C disks at 1.3 x 10(7) cells/cm3 (low density) or 7.6 x 10(7) cells/cm3 (high density). Constructs were directly implanted subcutaneously in nude mice for up to 8 weeks or precultured for 2 weeks before implantation. Preculture medium contained either transforming growth factor-beta1 (TGF-beta1, 1 ng/mL), fibroblast growth factor-2, and platelet-derived growth factor (proliferating medium) or TGF-beta1 (10 ng/mL) and insulin (differentiating medium). Both in vitro and after in vivo implantation, constructs derived by cell seeding at high versus low density and precultured in differentiating versus proliferating medium generated more cartilaginous tissues containing higher amounts of glycosaminoglycan and collagen type II and lower amounts of collagen type I, and with higher equilibrium moduli. As compared with direct implantation of freshly seeded scaffolds, preculture of AHAC-Hyalograft C constructs in differentiating medium, but not in proliferating medium, supported enhanced in vivo development of engineered cartilage. The effect of preculture was more pronounced when constructs were seeded at low density as compared with high density. This study indicates that preculture of human engineered cartilage in differentiating medium has the potential to provide grafts with higher equilibrium moduli and enhanced in vivo developmental capacity than freshly seeded scaffolds. These findings need to be validated in an orthotopic model system.
Transplantation of cultured chondrocytes can regenerate cartilage tissue in cartilage defects. This method requires serial cell passages to expand chondrocytes to a large number of cells for transplantation. However, as chondrocytes are expanded in number in monolayer culture, the cells gradually lose their differentiated phenotype and may not form cartilage tissue. This study investigated whether chondrocytes cultured through various passages maintain their potential to reexpress a chondrogenic phenotype in three-dimensional scaffolds and form cartilage tissue in vitro and in vivo. The growth rate, viability, synthesis of collagen type I and II, and apoptotic activity of chondrocytes with passage number of 1, 2 and 5 were compared during in vitro culture. As the passage number increased, the cell growth rate and viability decreased and apoptotic cell increased. Passage 2 chondrocytes exhibited a high expression of collagen type II and a low expression of collagen type I. In contrast, passage 5 chondrocytes exhibited a low expression of collagen type II and a high expression of collagen type I, indicating chondrocyte dedifferentiation. To examine the ability of chondrocytes to regenerate cartilage tissues in vitro and in vivo, chondrocytes were expanded in vitro to passage number of 1 or 5, seeded onto biodegradable polymer scaffolds, and maintained in vitro or implanted into subcutaneous spaces of athymic mice for 1 month. Histological and immunohistochemical analyses of cartilage tissues engineered in vitro and in vivo with passage 1 chondrocytes showed mature and well-formed cartilage and the presence of highly sulfated glycosaminoglycans and type II collagen, a collagen type produced by differentiated chondrocytes. In contrast, tissues engineered in vitro and in vivo with passage 5 chondrocytes did not have chondrocyte morphology or cartilage-specific extracellular matrices (i.e., glycosaminoglycans and type II collagen). The results of this study show that chondrocyte passage number is an important factor affecting the quality of cartilage tissue-engineered with the chondrocytes, and that chondrocytes.
Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes acquaintance with the principles and practice of PCR, as outlined in, for example, refs. 1, 2, 3, 4.
Objective
To establish a model and associated molecular markers for monitoring the capacity of in vitro–expanded chondrocytes to generate stable cartilage in vivo.
Methods
Adult human articular chondrocytes (AHAC) were prepared by collagenase digestion of samples obtained postmortem and were expanded in monolayer. Upon passaging, aliquots of chondrocyte suspensions were either injected intramuscularly into nude mice, cultured in agarose, or used for gene expression analysis. Cartilage formation in vivo was documented by histology, histochemistry, immunofluorescence for type II collagen, and proteoglycan analysis by ³⁵S‐sulfate incorporation and molecular sieve chromatography of the radiolabeled macromolecules. In situ hybridization for species‐specific genomic repeats was used to discriminate human‐derived from mouse‐derived cells. Gene expression dynamics were analyzed by semiquantitative reverse transcription–polymerase chain reaction.
Results
Intramuscular injection of freshly isolated AHAC into nude mice resulted in stable cartilage implants that were resistant to mineralization, vascular invasion, and replacement by bone. In vitro expansion of AHAC resulted in the loss of in vivo cartilage formation. This capacity was positively associated with the expression of fibroblast growth factor receptor 3, bone morphogenetic protein 2, and α1(II) collagen (COL2A1), and its loss was marked by the up‐regulation of activin receptor–like kinase 1 messenger RNA. Anchorage‐independent growth and the reexpression of COL2A1 in agarose culture were insufficient to predict cartilage formation in vivo.
Conclusion
AHAC have a finite capacity to form stable cartilage in vivo; this capacity is lost throughout passaging and can be monitored using a nude mouse model and associated molecular markers. This cartilage‐forming ability in vivo may be pivotal for successful cell‐based joint surface defect repair protocols.
... Thomas D Schmittgen 1 & Kenneth J Livak 2 . ABSTRACT. ... N. Engl. J . Med. ... 32, e178 (2004). | Article | PubMed | ChemPort |; Livak , KJ & Schmittgen , TD Analysis of relative gene expression data using real - time quantitative PCR and the 2 (- Delta Delta C(T)) Method . ...
... Thomas D Schmittgen 1 & Kenneth J Livak 2 . ABSTRACT. ... N. Engl. J . Med. ... 32, e178 (2004). | Article | PubMed | ChemPort |; Livak , KJ & Schmittgen , TD Analysis of relative gene expression data using real - time quantitative PCR and the 2 (- Delta Delta C(T)) Method . ...
Two different methods of presenting quantitative gene expression exist: absolute and relative quantification. Absolute quantification calculates the copy number of the gene usually by relating the PCR signal to a standard curve. Relative gene expression presents the data of the gene of interest relative to some calibrator or internal control gene. A widely used method to present relative gene expression is the comparative CT method also referred to as the 2−ΔΔCT method. This protocol provides an overview of the comparative CT method for quantitative gene expression studies. Also presented here are various examples to present quantitative gene expression data using this method.
Objective
To establish a model and associated molecular markers for monitoring the capacity of in vitro–expanded chondrocytes to generate stable cartilage in vivo.Methods
Adult human articular chondrocytes (AHAC) were prepared by collagenase digestion of samples obtained postmortem and were expanded in monolayer. Upon passaging, aliquots of chondrocyte suspensions were either injected intramuscularly into nude mice, cultured in agarose, or used for gene expression analysis. Cartilage formation in vivo was documented by histology, histochemistry, immunofluorescence for type II collagen, and proteoglycan analysis by 35S-sulfate incorporation and molecular sieve chromatography of the radiolabeled macromolecules. In situ hybridization for species-specific genomic repeats was used to discriminate human-derived from mouse-derived cells. Gene expression dynamics were analyzed by semiquantitative reverse transcription–polymerase chain reaction.ResultsIntramuscular injection of freshly isolated AHAC into nude mice resulted in stable cartilage implants that were resistant to mineralization, vascular invasion, and replacement by bone. In vitro expansion of AHAC resulted in the loss of in vivo cartilage formation. This capacity was positively associated with the expression of fibroblast growth factor receptor 3, bone morphogenetic protein 2, and α1(II) collagen (COL2A1), and its loss was marked by the up-regulation of activin receptor–like kinase 1 messenger RNA. Anchorage-independent growth and the reexpression of COL2A1 in agarose culture were insufficient to predict cartilage formation in vivo.ConclusionAHAC have a finite capacity to form stable cartilage in vivo; this capacity is lost throughout passaging and can be monitored using a nude mouse model and associated molecular markers. This cartilage-forming ability in vivo may be pivotal for successful cell-based joint surface defect repair protocols.
Designing PCR and sequencing primers are essential activities for molecular biologists around the world. This chapter assumes
acquaintance with the principles and practice of PCR, as outlined in, for example, refs.
1–4.
Joint pain due to cartilage degeneration is a serious problem, affecting people of all ages. Although many techniques, often surgical, are currently employed to treat this affliction, none have had complete success. Recent advances in biology and materials science have pushed tissue engineering to the forefront of new cartilage repair techniques. This review seeks to condense information for the biomaterialist interested in developing materials for this application. Articular cartilage anatomy, types of injury, and current repair methods are explained. The need for biomaterials, current commonly used materials for tissue-engineered cartilage, and considerations in scale-up of cell–biomaterial constructs are summarized.
Articular cartilage defects of the knee present diagnostic and treatment challenges for orthopaedic surgeons. As new data and technologies become available, treatment algorithms are continually being refined. It is important to examine treatment recommendations from the current literature and understand surgical techniques for articular cartilage repair.
Scaffold architecture and composition are important parameters in cartilage tissue engineering. In this in vitro study, we compared the morphology of four different cell-graft systems applied in clinical cartilage regeneration and analyzed the cell distribution (DAPI nuclei staining) and cell-scaffold interaction (SEM, TEM). Our investigations revealed major differences in cell distribution related to scaffold density, pore size and architecture. Material composition influenced the quantity of autogenous matrix used for cellular adhesion. Cell bonding was further influenced by the geometry of the scaffold subunits. On scaffolds with widely spaced fibers and a thickness less than the cell diameter, chondrocytes surrounded the scaffold fibers with cell extensions. On those fibers, chondrocytes were spherical, suggesting a differentiated phenotype. Fiber sizes smaller than chondrocyte size, and widely spaced, are therefore beneficial in terms of improved adhesion by cell shape adaptation. They also support the differentiated stage of chondrocytes by preventing the fibroblast-like and polygonal cell shape, at least briefly.
Treatments for managing articular cartilage defects of the knee, including drilling and abrasion arthroplasty, are not always effective. When they are, long-term benefits may not be maintained and osteoarthritis may develop. An alternative is autologous chondrocyte implantation (ACI), the surgical implantation of healthy cartilage cells into the damaged areas.
To determine the efficacy and safety of ACI in people with full thickness articular cartilage defects of the knee.
We searched the Cochrane Bone, Joint and Muscle Trauma Group Specialised Register (3 December 2008), the Cochrane Central Register of Controlled Trials (The Cochrane Library 2008, Issue 4), MEDLINE (1950 to November Week 3 2008), EMBASE (1980 to Week 48 2008), SPORTDiscus (3 December 2008), the WHO International Clinical Trials Registry Platform (4 December 2008), and Current Controlled Trials (3 December 2008).
Randomised and quasi-randomised trials comparing ACI with any other type of treatment (including no treatment or placebo) for symptomatic cartilage defects of the medial or lateral femoral condyle, femoral trochlea or patella.
Review authors selected studies for inclusion independently. We assessed risk of bias based on adequacy of the randomisation and allocation concealment process, potential for selection bias after allocation and level of masking. We did not pool data due to clinical and methodological heterogeneity.
Six heterogeneous trials were identified with 431 participants. Methodological flaws of the included trials included incomplete follow-up and inadequate reporting of outcomes. Three trials compared ACI versus mosaicplasty. One reported statistically significant results in favour of ACI at one year in the numbers of people with 'good' or 'excellent' functional results. Conversely, another trial found significant improvement for the mosaicplasty group when assessed using one functional scoring system at two years, but no statistically significant differences based on two other scoring systems. A third trial found no difference between ACI and mosaicplasty, 10 months on average after the surgery.There was no statistically significant difference in functional outcomes at two years in single trials comparing ACI with microfracture or characterised chondrocyte implantation versus microfracture. The results of the sixth trial comparing matrix-guided ACI versus microfracture were undermined by the severe loss to follow-up.
There is insufficient evidence to draw conclusions on the use of ACI for treating full thickness articular cartilage defects in the knee. Further good quality randomised controlled trials with long-term functional outcomes are required.
In autologous chondrocyte implantation (ACI), the periosteum patch which is sutured over the cartilage defect has been identified as a major source of complications such as periosteal hypertrophy. In the present retrospective study, we compared midterm results of first-generation ACI with a periosteal patch to second generation ACI using a biodegradable collagen fleece (BioSeed-C) in 82 patients suffering from chronic posttraumatic and degenerative cartilage lesions of the knee.
Clinical outcome was assessed in 42 patients of group 1 and in 40 patients of group 2 before implantation of the autologous chondrocytes and at a minimum follow-up of 2 years using the ICRS score, the modified Cincinnati score and the Lysholm score.
Although patients treated with BioSeed-C had more previous surgical procedures on their respective knees, highly significant improvements (P < 0.001) were assessed in both groups at comparable outcome levels: the ICRS score improved from grade D (poor) preoperatively to grade C (fair); the modified Cincinnati knee score from 3.26 to 6.4 (group 1) and 3.3 and 6.88 (group 2). Lysholm score improved from 33 to 70 points (group 1) and from 47 to 78 points (group 2), respectively. Revision surgery was due to symptomatic periosteal hypertrophy (n = 4), graft failure (n = 3), plica syndrome (n = 2) synovectomy (n = 1) (group 1); and graft failure (n = 2), debridement (n = 1), synovectomy (n = 2) (group 2).
These results suggest that BioSeed-C is an equally effective treatment option for focal degenerative chondral lesions of the knee in this challenging and complex patient profile.
The healing potential of damaged articular cartilage is limited. The NeoCart is a tissue-engineered collagen matrix seeded with autogenous chondrocytes designed for the repair of hyaline articular cartilage.
The NeoCart implant is well tolerated in the human knee.
Case series; Level of evidence, 4.
Eight patients (treatment group) with full-thickness cartilage injury were treated with the NeoCart and evaluated prospectively. Autogenous chondrocytes provided by arthroscopic biopsy were seeded into a 3-dimensional type I collagen scaffold. The seeded scaffold was subjected to a tissue-engineering protocol including treatment with a bioreactor. Implantation of the prepared cartilage tissue patch was performed via miniarthrotomy and secured with a collagen bioadhesive. Evaluations through 24 months postoperatively included the subjective International Knee Documentation Committee questionnaire, visual analog scale, range of motion, and cartilage-sensitive magnetic resonance imaging (MRI), including quantitative T2 mapping.
Pain scores after NeoCart implantation were significantly lower than baseline at 12 and 24 months after the procedure (P < .05). Improved function and motion were also noted at 24 months. Six patients had 67% to 100% defect fill at 24 months with MRI evaluation. One patient had moderate (33%-66%) defect fill, and another patient had poor (less than 33%) defect fill. Partial stratification of T2 values was observed for 2 patients at 12 months and 4 patients at 24 months. No patients experienced arthrofibrosis or implant hypertrophy.
Pain was significantly reduced 12 and 24 months after NeoCart treatment. Trends toward improved function and motion were observed 24 months after implantation. The MRI indicated implant stability and peripheral integration, defect fill without overgrowth, progressive maturation, and more organized cartilage formation.
Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n=32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.
Autologous chondrocyte transplantation/implantation (ACT/ACI) is becoming increasingly common for the treatment of large cartilage defects in the knee joint. The traditional ACT technique involves injection of a suspension of cells into the cartilage defect, which is covered with a periosteal flap or collagen membrane. The technique requires extensive suturing to create an effective seal; however, cell leakage remains a potential problem. Matrix-induced autologous chondrocyte implantation (MACI/MACT) avoids this potential problem by using a membrane on which chondrocytes are seeded and cultured for several days, before the membrane is cut to the correct size and shape of the defect. Time-consuming extensive suturing is unnecessary. However, cutting and repeated manipulation of the seeded membrane may result in the loss of critical chondrocytes. A modified technique termed ACT-collagen membrane seeding (ACT-Cs) has been developed in which expanded chondrocytes are applied to the collagen membrane after it has been cut to size, substantially reducing the risk of viable cell loss while retaining the ease and speed of the MACI/MACT procedure. In addition, the seeding of mitotically active chondrocytes onto the membrane after expansion and immediately before transplantation allows direct application of high cell concentrations.
Minced articular cartilage procedures are attractive surgical approaches for repairing articular cartilage, as they are 1-staged, autologous, and inserted on a carrier that can potentially be placed arthroscopically. The principle of mincing the autologous donor cartilage is to create a larger surface area for cartilage expansion. Placement on a scaffold carrier allows for a chondro-inductive and chondro-conductive milieu. Early animal and preclinical models have demonstrated hyaline-like tissue repair. Further work needs to be conducted in this promising approach.
The cultures of rabbit chondrocytes embedded in collagen gels were conducted to investigate the cell behaviors and consequent architectures of cell aggregation in an early culture phase. The chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) underwent a transition to spindle-shaped morphology, and formed the loose aggregates with a starburst shape by means of possible migration and gathering. These aggregates accompanied the poor production of collagen type II, while the cells seeded at 1.6 x 10(6) cells/cm(3) exhibited active proliferation to form the dense aggregates rich in collagen type II. Stereoscopic observation was performed at 5 days to define the migrating cells in terms of a morphology-relating parameter of sphericity determined for individual cells in the gels. The frequency of migrating cells decreased with increasing seeding density, while the frequency of dividing cells showed the counter trend. The culture seeded at 1.0 x 10(5) cells/cm(3) gave the migrating cell frequency of 0.25, the value of which was 25 times higher than that at 1.6 x 10(6) cells/cm(3). In addition, the analysis of mRNA expression revealed that the chondrocyte cells seeded at 1.0 x 10(5) cells/cm(3) showed appreciable down-regulation in collagen type II relating to differentiation and up-regulation in matrix metalloproteinases relating to migration, as compared to the cells seeded at 1.6 x 10(6) cells/cm(3). These data supports the morphological analyses concerning the cell migration and aggregate formation in the cultures with varied seeding densities. It is concluded that the seeding density is an important factor to affect the cell behaviors and architecture of aggregates and thereby to modulate the quality of cultured cartilage.
In vitro multiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generate neo-cartilage, but is known to induce cell-dedifferentiation. The aim of this study was to investigate whether multiplied chondrocytes can be used to generate neo-cartilage in vivo. Adult bovine articular chondrocytes, of various differentiation stages, were suspended in alginate at densities of 10 or 50 million/ml, either directly after isolation (P0) or after multiplication in monolayer for one (P1) or three passages (P3). Alginate with cells was seeded in demineralized bovine bone matrix (DBM) or a fleece of polylactic/polyglycolic acid (E210) and implanted in nude mice for 8 weeks. The newly formed tissue was evaluated by Alcian Blue and immunohistochemical staining for collagen type-II and type-I. Structural homogeneity of the tissue, composed of freshly isolated as well as serially passaged cells, was found to be enhanced by high-density seeding (50 million/ml) and the use of E210 as a carrier. The percentage of collagen type-II positive staining P3-cells was generally higher when E210 was used as a carrier. Furthermore, seeding P3-chondrocytes at the highest density (50 million/ml) enhanced collagen type-II expression. This study shows promising possibilities to generate structurally regular neo-cartilage using multiplied chondrocytes in alginate in combination with a fleece of polylactic/polyglycolic acid.
The healing of articular cartilage defects may be improved by the use of implantable three-dimensional matrices. The present study investigated the effects of four cross-linking methods on the compressive stiffness of collagen-glycosaminoglycan (CG) matrices and the interaction between adult canine articular chondrocytes and the matrix: dehydrothermal treatment (DHT), ultraviolet irradiation (UV), glutaraldehyde treatment (GTA), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC). The degree and kinetics of chondrocyte-mediated contraction, chondrocyte proliferation, and protein and glycosaminoglycan synthesis were evaluated over a four-week period in vitro. Cell-mediated contraction of the matrices varied with cross-linking: the most compliant DHT and UV matrices contracted the most (60% reduction in matrix diameter) and stiffest EDAC matrices contracted the least (30% reduction in matrix diameter). All cross-linking protocols permitted cell proliferation and matrix synthesis as measured by DNA content and radiolabeled sulfate and proline incorporation, respectively. During the first week in culture, a lower level of proliferation was seen in the GTA matrices but over the four-week culture period, the GTA and EDAC matrices provided for the greatest cell proliferation. On day 2, there was a significantly lower rate of 3H-proline incorporation in the GTA matrices (p<0.003) although at later time points, the EDAC and GTA matrices exhibited the highest levels of matrix synthesis. With regard to cartilage-specific matrix molecule synthesis, immunohistochemistry revealed a greater amount of type II collagen in DHT and UV matrices at the early time points. These findings serve as a foundation for future studies of tissue engineering of articular cartilage and the association of chondrocyte contraction and the processes of mitosis and biosynthesis.
The aim of the present study was the investigation of differential gene expression in primary human articular chondrocytes (HACs) and in cultivated cells derived from HACs.
Primary human articular chondrocytes (HACs) isolated from non-arthritic human articular cartilage and monolayer cultures of HACs were investigated by immunohistochemistry, Northern analysis, RT-PCR and cDNA arrays.
By immunohistochemistry we detected expression of collagen II, protein S-100, chondroitin-4-sulphate and vimentin in freshly isolated HACs. Cultivated HACs, however, showed only collagen I and vimentin expression. These data were corroborated by the results of Northern analysis using specifc cDNA probes for collagens I, II and III and chondromodulin, respectively, demonstrating collagen II and chondromodulin expression in primary HACs but not in cultivated cells. Hybridization of mRNA from primary HACs and cultivated cells to cDNA arrays revealed additional transcriptional changes associated with dedifferentiation during propagation of chondrocytes in vitro. We found a more complex hybridization pattern for primary HACs than for cultivated cells. Of the genes expressed in primary HACs the early growth response (EGR1) transcription factor showed the strongest expression whereas D-type cyclin was expressed in proliferating cells. Other factors associated with differentiated HACs were the adhesion molecules ICAM-1 and VCAM-1, VEGF, TGFbeta2, and the monocyte chemotactic protein receptor.
Our data support the hypothesis that HACs dedifferentiate when grown in monolayer cultures. Moreover, the expression patterns also show that proliferation and differentiation are exclusive features of human chondrocytes.
Data pertaining to the quantitative structural features and organization of normal articular cartilage are of great importance in understanding its biomechanical properties and in attempting to establish this tissue's counterpart by engineering in vitro. A comprehensive set of such baseline data is, however, not available for humans. It was the purpose of the present study to furnish the necessary information.
The articular cartilage layer covering the medial femoral condyle of deceased persons aged between 23 and 49 years was chosen for the morphometric analysis of cell parameters using confocal microscopy in conjunction with unbiased stereological methods. The height of the hyaline articular cartilage layer, as well as that of the calcified cartilage layer and the subchondral bone plate, were also measured.
The mean height of the hyaline articular cartilage layer was found to be 2.4mm, the volume density of chondrocytes therein being 1.65%, the number of cells per mm(3) of tissue 9626 and the mean cell diameter 13 microm. Other estimators (including matrix mass per cell and cell profile density) were also determined.
A comparison of these normal human quantitative data with those published for experimental animals commonly used in orthopaedic research reveals substantial differences, consideration of which in tissue engineering strategies destined for human application are of paramount importance for successful repair.
Animal models are widely used to develop and evaluate tissue-engineering techniques for the reconstruction of damaged human articular cartilage. For the purpose of this review, these model systems will include in vitro culture of animal cells and explants, heterotopic models of chondrogenesis, and articular cartilage defect models. The objectives for these preclinical studies are to engineer articular cartilage for the functional restoration of a joint surface that appears anatomically, histologically, biologically, biochemically, and mechanically to resemble the original joint surface. While no animal model permits direct application to humans, each is capable of yielding principles on which decisions can be made that might eventually translate into a human application. Clearly, the use of animal models has and will continue to play a significant role in the advancement of this field. Each animal model has specific advantages and disadvantages. The key issue in the selection of an appropriate animal model is to match the model to the question being investigated and the hypothesis to be tested. The purpose of this review is to discuss issues regarding animal model selection, the benefits and limitations of these model systems, scaffold selection with emphasis on polymers, and evaluation of the tissue-engineered articular cartilage.
Using a previously established canine model for repair of articular cartilage defects, this study evaluated the 15-week healing of chondral defects (i.e., to the tidemark) implanted with an autologous articular chondrocyte-seeded type II collagen scaffold that had been cultured in vitro for four weeks prior to implantation. The amount and composition of the reparative tissue were compared to results from our prior studies using the same animal model in which the following groups were analyzed: defects implanted with autologous chondrocyte-seeded collagen scaffolds that had been cultured in vitro for approximately 12 h prior to implantation, defects implanted with autologous chondrocytes alone, and untreated defects. Chondrocytes, isolated from articular cartilage harvested from the left knee joint of six adult canines, were expanded in number in monolayer for three weeks, seeded into porous type II collagen scaffolds, cultured for an additional four weeks in vitro and then implanted into chondral defects in the trochlear groove of the right knee joints. The percentages of specific tissue types filling the defects were evaluated histomorphometrically and certain mechanical properties of the repair tissue were determined. The reparative tissue filled 88+/-6% (mean+/-SEM; range 70-100%) of the cross-sectional area of the original defect, with hyaline cartilage accounting for 42+/-10% (range 7-67%) of defect area. These values were greater than those reported previously for untreated defects and defects implanted with a type II collagen scaffold seeded with autologous chondrocytes within 12 h prior to implantation. Most striking, was the decreased amount of fibrous tissue filling the defects in the current study, 5+/-5% (range 0-26%) as compared to previous treatments. Despite this improvement, indentation testing of the repair tissue formed in this study revealed that the compressive stiffness of the repair tissue was well below (20-fold lower stiffness) that of native articular cartilage.
Articular chondrocytes are often expanded in vitro and then used to assist in healing articular cartilage defects. We investigated the extent of dedifferentiation in monolayer-passaged, zonal articular chondrocytes by using quantitative, real-time PCR. The relative gene expressions for collagen type I and II, aggrecan, and superficial zone protein were analyzed for relevant passage numbers (P0-P4) to determine how the expansion of chondrocytes affects the expression of cartilage extracellular matrix proteins. Results reveal that dramatic changes occur as early as first passage. Furthermore, these changes are shown to persist even when the expanded cells are encapsulated in 3D, alginate beads. Successful tissue engineering and autologous cell transplantation procedures rely heavily on having a cell source that expresses the chondrocytic phenotype. The results of this study suggest that major problems exist at the front-end of cartilage regeneration efforts.
Understanding of articular cartilage physiology, remodelling mechanisms, and evaluation of tissue engineering repair methods requires reference information regarding normal structural organization. Our goals were to examine the variation of cartilage cell and matrix morphology in different topographical areas of the adult human knee joint.
Osteochondral explants were acquired from seven distinct anatomical locations of the knee joints of deceased persons aged 20-40 years and prepared for analysis of cell, matrix and tissue morphology using confocal microscopy and unbiased stereological methods. Differences between locations were identified by statistical analysis.
Medial femoral condyle cartilage had relatively high cell surface area per unit tissue volume in the superficial zone. In the transitional zone, meniscus-covered lateral tibia cartilage showed elevated chondrocyte densities compared to the rest of the knee while lateral femoral condyle cartilage exhibited particularly large chondrocytes. Statistical analyses indicated highly uniform morphology throughout the radial zone (lower 80% of cartilage thickness) in the knee, and strong similarities in cell and matrix morphologies among cartilage from the femoral condyles and also in the mediocentral tibial plateau. Throughout the adult human knee, the mean matrix volume per chondron was remarkably constant at approximately 224,000 microm(3), corresponding to approximately 4.6 x 10(6) chondrons per cm(3).
The uniformity of matrix volume per chondron throughout the adult human knee suggests that cell-scale biophysical and metabolic constraints may place limitations on cartilage thickness, mechanical properties, and remodelling mechanisms. Data may also aid the evaluation of cartilage tissue engineering treatments in a site-specific manner. Results indicate that joint locations which perform similar biomechanical functions have similar cell and matrix morphologies; findings may therefore also provide clues to understanding conditions under which focal lesions leading to osteoarthritis may occur.
Osteochondral injury is therapeutically irreversible within current treatment parameters. Autologous chondrocyte implantation (ACI) promises to regenerate hyaline articular cartilage, but conventional ACI is plagued by complications determined by periosteal grafting. Here we propose the utilization of collagen membrane in ACI as an effective bioscaffold for the regeneration of osteochondral lesions. Using a rabbit model of osteochondral injury, we have inoculated autologous chondrocytes onto a type I/III collagen scaffold [so-called matrix-induced ACI (MACI)] and implanted into 3-mm osteochondral knee defects. All untreated defect histology showed inferior fibrocartilage and/or fibrous tissue repair. In our time-course study, ACI with type I/III collagen membrane regenerated cartilage with healthy osteochondral architecture in osteochondral defects at 6 weeks. At 12 weeks, articular cartilage regeneration was maintained, with reduced thickness and proteoglycan compared with the adjacent cartilage. Both 6-week (p < 0.01) and 12-week (p < 0.05) ACI with collagen membrane showed significant improvement as compared with untreated controls. To further examine the efficacy of cartilage regeneration by ACI, we conducted a dose-response study, using chondrocytes at various cell densities between 10(4) and 10(6) cells/cm(2). The results showed that cell density had no effect on outcome histology, but all cell densities were significantly better than untreated controls (p < 0.01) and cell-free collagen membrane treatment (p < 0.05). In short, our data suggest that autologous chondrocyte-seeded type I/III collagen membrane is an effective method for the treatment of focal osteochondral knee injury in rabbits.
Articular cartilage in adults has a limited capacity for self-repair after a substantial injury. Surgical therapeutic efforts to treat cartilage defects have focused on delivering new cells capable of chondrogenesis into the lesions. Autologous chondrocyte transplantation (ACT) is an advanced cell-based orthobiologic technology used for the treatment of chondral defects of the knee that has been in clinical use since 1987 and has been performed on 12,000 patients internationally. With ACT, good to excellent clinical results are seen in isolated post-traumatic lesions of the knee joint in the younger patient, with the formation of hyaline or hyaline-like repair tissue. In the classic ACT technique, chondrocytes are isolated from small slices of cartilage harvested arthroscopically from a minor weight-bearing area of the injured knee. The extracellular matrix is removed by enzymatic digestion, and the cells are then expanded in monolayer culture. Once a sufficient number of cells has been obtained, the chondrocytes are implanted into the cartilage defect, using a periosteal patch over the defect as a method of cell containment. The major complications are periosteal hypertrophy, delamination of the transplant, arthrofibrosis and transplant failure. Further improvements in tissue engineering have contributed to the next generation of ACT techniques, where cells are combined with resorbable biomaterials, as in matrix-associated autologous chondrocyte transplantation (MACT). These biomaterials secure the cells in the defect area and enhance their proliferation and differentiation.
Chondrogenesis is the earliest phase of skeletal development, involving mesenchymal cell recruitment and migration, condensation of progenitors, and chondrocyte differentiation, and maturation and resulting in the formation of cartilage and bone during endochondral ossification. This process is controlled exquisitely by cellular interactions with the surrounding matrix, growth and differentiation factors, and other environmental factors that initiate or suppress cellular signaling pathways and transcription of specific genes in a temporal-spatial manner. Vertebrate limb development is controlled by interacting patterning systems involving prominently the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and hedgehog pathways. Both positive and negative signaling kinases and transcription factors, such as Sox9 and Runx2, and interactions among them determine whether the differentiated chondrocytes remain within cartilage elements in articular joints or undergo hypertrophic maturation prior to ossification. The latter process requires extracellular matrix remodeling and vascularization controlled by mechanisms that are not understood completely. Recent work has revealed novel roles for mediators such as GADD45beta, transcription factors of the Dlx, bHLH, leucine zipper, and AP-1 families, and the Wnt/beta-catenin pathway that interact at different stages during chondrogenesis.
In recent years, a great variety of different matrix systems for the cultivation of chondrocytes have been developed. Although some of these scaffolds show promising experimental results in vitro, the potential clinical value remains unclear. In this comparative study, we propagated human articular chondrocytes precultivated in monolayer culture on six different scaffolds (collagen gels, membranes and sponges) under standardized in vitro conditions. Mechanical properties of the matrix systems were not improved significantly by cultivation of human chondrocytes under the given in vitro conditions. The gel systems (CaReS, Ars Artho, Germany and Atelocollagen, Koken, Japan) showed a homogeneous cell distribution; chondrocytes propagated on Chondro-Gide (Geistlich Biomaterials, Switzerland) and Integra membranes (Integra, USA) were building multilayers. Only few cells penetrated the two Atelocollagen honeycomb sponges (Koken, Japan). During cultivation, chondrocytes propagated on all systems showed a partial morphological redifferentiation, which was best with regard to the gel systems. In general, only small amounts of collagen type-II protein could be detected in the pericellular region and chondrocytes failed to build a territorial matrix. During the first two weeks of cultivation, the two gel systems showed a significantly higher collagen type-II gene expression and a lower collagen type-I gene expression than the other investigated matrix systems. Although collagen gels seem to be superior when dealing with deep cartilage defects, membrane systems might rather be useful in improving conventional autologous chondrocyte transplantation or in combination with gel systems.
This study investigated the effect of unidirectional and multidirectional motion patterns on gene expression and molecule release of chondrocyte-seeded 3D scaffolds. Resorbable porous polyurethane scaffolds were seeded with bovine articular chondrocytes and exposed to dynamic compression, applied with a ceramic hip ball, alone (group 1), with superimposed rotation of the scaffold around its cylindrical axis (group 2), oscillation of the ball over the scaffold surface (group 3), or oscillation of ball and scaffold in phase difference (group 4). Compared with group 1, the proteoglycan 4 (PRG4) and cartilage oligomeric matrix protein (COMP) mRNA expression levels were markedly increased by ball oscillation (groups 3 and 4). Furthermore, the collagen type II mRNA expression was enhanced in the groups 3 and 4, while the aggrecan and tissue inhibitor of metalloproteinase-3 (TIMP-3) mRNA expression levels were upregulated by multidirectional articular motion (group 4). Ball oscillation (groups 3 and 4) also increased the release of PRG4, COMP, and hyaluronan (HA) into the culture media. This indicates that the applied stimuli can contribute to the maintenance of the chondrocytic phenotype of the cells. The mechanical effects causing cell stimulation by applied surface motion might be related to fluid film buildup and/or frictional shear at the scaffold-ball interface. It is suggested that the oscillating ball drags the fluid into the joint space, thereby causing biophysical effects similar to those of fluid flow.
To examine a retroviral gene transfer to chondrocytes in vitro and in vivo in tissue-engineered cell-collagen constructs articular chondrocytes from rabbits and humans were isolated and transduced with VSV.G pseudotyped murine leukemia virus-derived retroviral vectors. Viral supernatants were generated by transient transfection of 293T cells using the pBullet retroviral vector carrying the nlslacZ gene, a Moloney murine leukemia virus gag/pol plasmid and a VSV.G coding plasmid. Transduction efficiency was analyzed by fluorescence-activated-cell-sorter analysis and transduced autologous chondrocytes from rabbits were seeded on collagen-scaffolds and implanted into osteochondral defects in the patellar groove of the rabbit's femur (n=10). LacZ-expression was analyzed by X-gal staining on total knee explants and histological sections. Retroviral transduction efficiency exceeded 92.3% (SEM+/-3.5%) in rabbit articular chondrocytes, 74.7% (SEM+/-1.8%) in human articular chondrocytes and 52.7% (SEM+/-5.8%) in osteoarthritic human chondrocytes. Reporter gene expression remained high after 15 weeks in 75.7% (SEM+/-8.2%) of transduced rabbit articular chondrocytes. In vivo, intraarticular beta-galactosidase activity could be determined in the majority of implanted chondrocytes in the osteochondral defects after 4 weeks.