Expanded phenotypically stable chondrocytes persist in the repair tissue and contribute to cartilage matrix formation and structural integration in a goat model of autologous chondrocyte implantation.

Autologous chondrocyte implantation (ACI) is an established technique to repair joint surface defects. Although there is some indirect evidence that the expanded chondrocytes are required to achieve proper healing, the role they play in the repair process is not clear yet. To monitor the persistence and the phenotype of the injected chondrocytes in the repair tissue (RT) we have optimized a fluorescent labeling protocol for articular chondrocytes, which allows cell tracking in vivo for up to 14 weeks, using the fluorescent dye PKH26. We have combined in vivo cell tracking, with the immune-detection of collagen type II protein in a goat model of ACI. Our data indicate that the implanted cells can persist for at least 14 weeks in the defects, can participate in the integration with the surrounding tissues, and become structural part of the RT, rich in collagen type II and sulfated proteoglycans. Albeit with a small number of samples, our data provide proof of principal that the implanted chondrocytes can contribute to structural cartilage repair in a goat model of ACI.     

Histologic analysis of tissue after failed cartilage repair procedures

This study evaluated the composition of reparative tissue retrieved during revision surgery from full thickness chondral defects in 18 patients in whom abrasion arthroplasty (n = 12), grafting of perichondrial flaps (n = 4), and periosteal patching augmented by autologous chondrocyte implantation in cell suspension (n = 6) failed to provide lasting relief of symptoms. The defects were graded by gross appearance, and all of the tissue filling the defect was retrieved. Histologic evaluation included histomorphometric analysis of the percentage of selected tissue types in cross sections. Immunohistochemistry was performed using antibodies to Types I, II, and X collagen. The histologic appearance of material retrieved after abrasion arthroplasty was that of fibrous, spongiform tissue comprising Type I collagen in 22% +/- 9% (mean +/- standard error of the mean) of the cross sectional area, and degenerating hyaline tissue (30% +/- 10%) and fibrocartilage (28% +/- 7%) with positive Type II collagen staining. Three of four specimens obtained after implantation of perichondrium failed as a result of bone formation that was found in 19% +/- 6% of the cross sectional area, including areas staining positive for Type X collagen, as an indicator for hypertrophic chondrocytes. Revision after autologous chondrocyte implantation was associated with partial displacement of the periosteal graft from the defect site because of insufficient ongrowth or early suture failure. When the graft edge displaced, repair tissue was fibrous (55% +/- 11%), whereas graft tissue attached to subchondral bone displayed hyaline tissue (to 6%) and fibrocartilage (to 12%) comprising Type II collagen at 3 months after surgery. Evaluation of retrieved repair tissue after selected cartilage repair procedures revealed distinctive histologic features reflecting the mechanisms of failure.     

Paracrine effect of transplanted rib chondrocyte spheroids supports formation of secondary cartilage repair tissue

The study's objective was to investigate if transplanted chondrocyte or periosteal cell spheroids have influence on ingrowing bone marrow‐derived cells in a novel cartilage repair approach in miniature pigs. Autologous rib chondrocytes or periosteal cells were cultured as spheroids and press‐fitted into cavities that were milled into large, superficial chondral lesions of the patellar joint surface. Within the milled cavities, the subchondral bone plate was either penetrated or left intact (full‐thickness or partial‐thickness cavities). The transplantation of chondrocyte spheroids into full‐thickness cavities induced the formation of additional secondary repair cartilage that exceeded the original volume of the transplanted spheroids. The resulting continuous tissue was rich in proteoglycans and stained positive for type II collagen. Cell labeling revealed that secondarily invading repair cells did not originate from transplanted spheroids, but rather from arroded bone marrow. However, secondary invasion of repair cells was less pronounced following transplantation of periosteal cells and absent in partial‐thickness cavities. According to in vitro analyses, these observations could be ascribed to the ability of chondrocyte spheroids to secrete relevant amounts of bone morphogenetic protein‐2, which was not detected for periosteal cells. Transplanted chondrocyte spheroids exert a dual function: they provide cells for the repair tissue and have a stimulatory paracrine activity, which promotes ingrowth and chondrogenesis of bone marrow‐derived cells. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res     

Genetic modification of chondrocytes with insulin-like growth factor-1 enhances cartilage healing in an equine model

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 x 10(7) AdIGF-1 modified chondrocytes and the contralateral joint received 2 x 10(7) naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation.     

Articular cartilage repair with recombinant human type II collagen/polylactide scaffold in a preliminary porcine study

The purpose of this study was to investigate the potential of a novel recombinant human type II collagen/polylactide scaffold (rhCo‐PLA) in the repair of full‐thickness cartilage lesions with autologous chondrocyte implantation technique (ACI). The forming repair tissue was compared to spontaneous healing (spontaneous) and repair with a commercial porcine type I/III collagen membrane (pCo). Domestic pigs (4‐month‐old, n = 20) were randomized into three study groups and a circular full‐thickness chondral lesion with a diameter of 8 mm was created in the right medial femoral condyle. After 3 weeks, the chondral lesions were repaired with either rhCo‐PLA or pCo together with autologous chondrocytes, or the lesion was only debrided and left untreated for spontaneous repair. The repair tissue was evaluated 4 months after the second operation. Hyaline cartilage formed most frequently in the rhCo‐PLA treatment group. Biomechanically, there was a trend that both treatment groups resulted in better repair tissue than spontaneous healing. Adverse subchondral bone reactions developed less frequently in the spontaneous group (40%) and the rhCo‐PLA treated group (50%) than in the pCo control group (100%). However, no statistically significant differences were found between the groups. The novel rhCo‐PLA biomaterial showed promising results in this proof‐of‐concept study, but further studies will be needed in order to determine its effectiveness in articular cartilage repair. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:745–753, 2016.     

The use of debrided human articular cartilage for autologous chondrocyte implantation: maintenance of chondrocyte differentiation and proliferation in type I collagen gels.

Autologous chondrocyte implantation (ACI) is the most promising surgical treatment for large full thickness knee joint articular cartilage (AC) defects where cells from healthy non-weight bearing area AC are multiplied in vitro and implanted into such defects. In the routine surgical procedure for symptomatic knee full thickness AC defects, damaged AC surrounding the edge and the base of such defects is usually debrided and discarded. The purpose of this study was to examine if chondrocytes from this 'debrided' AC can proliferate, synthesize a cartilage specific matrix and thus can be used for ACI. METHODS: Biopsies were retrieved from 12 patients (debrided articular cartilage: DAC, aged 35-61) and from two autopsies (normal articular cartilage: NAC, aged 21 and 25). Chondrocytes were isolated, seeded at low density in type I collagen gels and as monolayer cultures for 4 weeks without passage. RESULTS: After 4 weeks cultures in type I collagen gels, cell proliferation from DAC (18.34 +/- 1.95 fold) was similar to cells from NAC (11.24 +/- 1.02 fold). Syntheses of proteoglycan and collagen in DAC were also similar to NAC. Newly synthesized matrices in gel cultures consisted predominantly of type II collagen as shown by immuno-labelling and SDS-PAGE followed by fluorography. Chondrocytes from 'debrided human AC' cultured at low density in type I collagen gels may be used for the ACI procedure as they provide sufficient viable cell numbers for ACI and maintain their chondrocyte phenotype as they synthesize a cartilage-like matrix.     

Sizable Scaffold‐Free Tissue‐Engineered Articular Cartilage Construct for Cartilage Defect Repair

This study introduces an implantable scaffold‐free cartilage tissue construct (SF) that is composed of chondrocytes and their self‐produced extracellular matrix (ECM). Chondrocytes were grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 21, and 35 days). For in vivo implantation, full‐thickness defects (n = 5) were manually created on the trochlear groove of the both knees of rabbits (16‐week old) and 3 week‐cultured SF construct was implanted as an allograft for a month. The left knee defects were implanted with 1, 7, and 21 days in vitro cultured scaffold‐free engineered cartilages. (group 2, 3, and 4, respectively). The maturity of the engineered cartilages was evaluated by histological, chemical and mechanical assays. The repair of damaged cartilages was also evaluated by gross images and histological observations at 4, 8, and 12 weeks postsurgery. Although defect of groups 1, 2, and 3 were repaired with fibrocartilage tissues, group 4 (21 days) showed hyaline cartilage in the histological observation. In particular, mature matrix and columnar organization of chondrocytes and highly expressed type II collagen were observed only in 21 days in vitro cultured SF cartilage (group 4) at 12 weeks. As a conclusion, cartilage repair with maturation was recapitulated when implanted the 21 day in vitro cultured scaffold‐free engineered cartilage. When implanting tissue‐engineered cartilage, the maturity of the cartilage tissue along with the cultivation period can affect the cartilage repair.     

Mechanical properties and structure–function relationships in articular cartilage repaired using IGF‐I gene‐enhanced chondrocytes

Several studies have demonstrated the benefits of IGF‐I gene therapy in enhancing the histologic and biochemical content of cartilage repaired by chondrocyte transplantation. However, there is little to no data on the mechanical performance of IGF‐I augmented cartilage grafts. This study evaluated the compressive properties of full‐thickness chondral defects in the equine femur repaired with and without IGF‐I gene therapy. Animals were randomly assigned to one of three study cohorts based on chondrocyte treatment provided in each defect: (i) IGF‐I gene delivered by recombinant adeno‐associated virus (rAAV)‐5; (ii) AAV‐5 delivering GFP as a reporter; (iii) naïve cells without virus. In each case, the opposite limb was implanted with a fibrin carrier without cells. Samples were prepared for confined compression testing to measure the aggregate modulus and hydraulic permeability. All treatment groups, regardless of cell content or transduction, had mechanical properties inferior to native cartilage. Overexpression of IGF‐I increased modulus and lowered permeability relative to other treatments. Investigation of structure–property relationships revealed that Ha and k were linearly correlated with GAG content but logarithmically correlated with collagen content. This provides evidence that IGF‐I gene therapy can improve healing of articular cartilage and can greatly increase the mechanical properties of repaired grafts. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:149–153, 2016.     

Rabbit articular cartilage defects treated by allogenic chondrocyte transplantation

Articular cartilage defects have a poor capacity for repair. Most of the current treatment options result in the formation of fibro-cartilage, which is functionally inferior to normal hyaline articular cartilage. We studied the effectiveness of allogenic chondrocyte transplantation for focal articular cartilage defects in rabbits. Chondrocytes were cultured in vitro from cartilage harvested from the knee joints of a New Zealand White rabbit. A 3 mm defect was created in the articular cartilage of both knees in other rabbits. The cultured allogenic chondrocytes were transplanted into the defect in the right knees and closed with a periosteal flap, while the defects in the left knees served as controls and were closed with a periosteal flap alone, without chondrocytes. Healing of the defects was assessed at 12 weeks by histological studies. Allogenic chondrocyte transplantation significantly increased the amount of newly formed repair tissue (P=0.04) compared with that found in the control knees. The histological quality score of the repair tissue was significantly better (P=0.05), with more hyaline characteristics in the knees treated with allogenic chondrocytes than in the control knees. Articular cartilage defects treated with allogenic chondrocyte transplantation result in better repair tissue formation with hyaline characteristics than those in control knees.     

New Technique for Cell-Seeded Collagen Matrix-Supported Autologous Chondrocyte Transplantation

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.     

Formation of cartilage repair tissue in articular cartilage defects pretreated with microfracture and covered with cell‐free polymer‐based implants

The aim of our study was to evaluate the mid‐term outcome of a cell‐free polymer‐based cartilage repair approach in a sheep cartilage defect model in comparison to microfracture treatment. Cell‐free, freeze‐dried implants (chondrotissue®) made of a poly‐glycolic acid (PGA) scaffold and hyaluronan were immersed in autologous serum and used for covering microfractured full‐thickness articular cartilage defects of the sheep (n = 4). Defects treated with microfracture only served as controls (n = 4). Six months after implantation, cartilage implants and controls were analyzed by immunohistochemical staining of type II collagen, histological staining of proteoglycans, and histological scoring. Histological analysis showed the formation of a cartilaginous repair tissue rich in proteoglycans. Histological scoring documented significant improvement of repair tissue formation when the defects were covered with the cell‐free implant, compared to controls treated with microfracture. Immunohistochemistry showed that the cell‐free implant induced cartilaginous repair tissue and type II collagen. Controls treated with microfracture showed marginal formation of a mixed‐type repair tissue consisting of cartilaginous tissue and fibro‐cartilage. Covering of microfractured defects with the cell‐free polymer‐based cartilage implant is suggested to be a promising treatment option for cartilage defects and improves the regeneration of articular cartilage. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1353–1360, 2009     

Evaluation of autologous chondrocyte transplantation via a collagen membrane in equine articular defects: results at 12 and 18 months

OBJECTIVE: To evaluate a technique of autologous chondrocyte implantation (ACI) similar to the other techniques using cell-seeded resorbable collagen membranes in large articular defects. METHODS: Autologous cartilage was harvested arthroscopically from the lateral trochlear ridge of the femur in fifteen 3-year-old horses. After culture and expansion of chondrocytes the newly created ACI construct (autologous chondrocytes cultured expanded, seeded on a collagen membrane, porcine small intestine submucosa) was implanted into 15mm defects on the medial trochlear ridge of the femur in the opposite femoropatellar joint. Using two defects in each horse, the ACI technique was compared to collagen membrane alone (CMA) and empty cartilage defects (ECDs). RESULTS: Arthroscopic evaluations at 4, 8, 12 and 18 months demonstrated that CMA was significantly worse compared to ACI or ECD treatments, with ACI having the best overall subjective grade. Overall raw histological scores demonstrated a significant improvement with ACI compared to either CMA or ECD treated defects and ACI defects had significantly more immunohistochemical staining for aggrecan than CMA or ECD treated defects (with significantly more type II collagen in ACI and ECD compared to CMA defects) at 12 and 18 months. CONCLUSIONS: Histologic and immunohistochemistry results from this long-term randomized study are particularly encouraging and demonstrate superiority with the ACI technique. Although there is no comparable study published with the traditional ACI technique in the horse (or with such a large defect size in another animal model), the use of a solid autologous cell-seeded-constructed implant would appear to offer considerable clinical advantages.     

Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7.

BACKGROUND: Cartilage has a limited capacity to heal. Although chondrocyte transplantation is a useful therapeutic strategy, the repair process can be lengthy. Previously we have shown that over expression of bone morphogenetic protein-7 (BMP-7) in chondrocytes by adenovirus-mediated gene transfer leads to increased matrix synthesis and cartilage-like tissue formation in vitro. In this context we hypothesized that implantation of genetically modified chondrocytes expressing BMP-7 would accelerate the formation of hyaline-like repair tissue in an equine model of cartilage defect repair. METHODS: Chondrocytes treated with adenovirus vector encoding BMP-7 (AdBMP-7) or as control, an adenovirus vector encoding an irrelevant gene (Escherichia coli cytosine deaminase, AdCD) were implanted into extensive (15 mm diameter) articular cartilage defects in the patellofemoral joints of 10 horses. Biopsies were performed to evaluate early healing at 4 weeks. At the terminal time point of 8 months, repairs were assessed for morphology, MRI appearance, compressive strength, biochemical composition and persistence of implanted cells. RESULTS: Four weeks after surgery AdBMP-7-treated repairs showed an increased level of BMP-7 expression and accelerated healing, with markedly more hyaline-like morphology than control. Quantitative real-time polymerase chain reaction (PCR) analysis of the repair tissue 8 months after surgery showed that few implanted cells persisted. By this time, the controls had healed similarly to the AdBMP-7-treated defects, and no difference was detected in the morphologic, biochemical or biomechanical properties of the repair tissues from the two treatment groups. CONCLUSIONS: Implantation of genetically modified chondrocytes expressing BMP-7 accelerates the appearance of hyaline-like repair tissue in experimental cartilage defects. CLINICAL RELEVANCE: Rehabilitation after cell-based cartilage repair can be prolonged, leading to decreased patient productivity and quality of life. This study shows the feasibility of using genetically modified chondrocytes to accelerate cartilage healing.     

Treatment of a Focal Articular Cartilage Defect of the Talus with Polymer-Based Autologous Chondrocyte Implantation: A 12-Year Follow-Up Period

Autologous chondrocyte implantation (ACI) is a first-line treatment option for large articular cartilage defects. Although well-established for cartilage defects in the knee, studies of the long-term outcomes of matrix-assisted ACI to treat cartilage defects in the ankle are rare. In the present report, we describe for the first time the long-term clinical and radiologic results 12 years after polymer-based matrix-assisted ACI treat a full-thickness talar cartilage defect in a 25-year-old male patient. The clinical outcome was assessed using the visual analog scale and Freiburg ankle score, magnetic resonance imaging evaluation using the Henderson-Kreuz scoring system and T2 mapping. Clinical assessment revealed improved visual analog scale and Freiburg ankle scores. The radiologic analysis and T2 relaxation time values indicated the formation of hyaline-like repair tissue. Polymer-based autologous chondrocytes has been shown to be a safe and clinically effective long-term treatment of articular cartilage defects in the talus.     

Enhanced early chondrogenesis in articular defects following arthroscopic mesenchymal stem cell implantation in an equine model.

Mesenchymal stem cells (MSCs) provide an important source of pluripotent cells for musculoskeletal tissue repair. This study examined the impact of MSC implantation on cartilage healing characteristics in a large animal model. Twelve full-thickness 15-mm cartilage lesions in the femoropatellar articulations of six young mature horses were repaired by injection of a self-polymerizing autogenous fibrin vehicle containing mesenchymal stem cells, or autogenous fibrin alone in control joints. Arthroscopic second look and defect biopsy was obtained at 30 days, and all animals were euthanized 8 months after repair. Cartilage repair tissue and surrounding cartilage were assessed by histology, histochemistry, collagen type I and type II immunohistochemistry, collagen type II in situ hybridization, and matrix biochemical assays. Arthroscopic scores for MSC-implanted defects were significantly improved at the 30-day arthroscopic assessment. Biopsy showed MSC-implanted defects contained increased fibrous tissue with several defects containing predominantly type II collagen. Long-term assessment revealed repair tissue filled grafted and control lesions at 8 months, with no significant difference between stem cell-treated and control defects. Collagen type II and proteoglycan content in MSC-implanted and control defects were similar. Mesenchymal stem cell grafts improved the early healing response, but did not significantly enhance the long-term histologic appearance or biochemical composition of full-thickness cartilage lesions.     

The treatment of osteochondral lesions using a combination of autologous chondrocyte implantation and autograft: three-year follow-up

In this study a combination of autologous chondrocyte implantation (ACI) and the osteochondral autograft transfer system (OATS) was used and evaluated as a treatment option for the repair of large areas of degenerative articular cartilage. We present the results at three years post-operatively. Osteochondral cores were used to restore the contour of articular cartilage in 13 patients with large lesions of the lateral femoral condyle (n = 5), medial femoral condyle (n = 7) and patella (n = 1). Autologous cultured chondrocytes were injected underneath a periosteal patch covering the cores. After one year, the patients had a significant improvement in their symptoms and after three years this level of improvement was maintained in ten of the 13 patients. Arthroscopic examination revealed that the osteochondral cores became well integrated with the surrounding cartilage. We conclude that the hybrid ACI/OATS technique provides a promising surgical approach for the treatment of patients with large degenerative osteochondral defects.     

Analysis of rabbit articular cartilage repair after chondrocyte implantation using optical coherence tomography

OBJECTIVE: To evaluate the utility and limitations of optical coherence tomography (OCT) for immediate, high-resolution structural analysis of rabbit articular repair tissue following chondrocyte implantation without excising or sectioning the specimen. METHODS: Full thickness articular cartilage defects were created in the patellar grooves of 30 adult rabbit knee joints. Allogenic cultured chondrocytes embedded in collagen gels were implanted into the surgical defects. A periosteal patch was then sutured over the chondrocyte-collagen composites. Six animals per time point were sacrificed at 2, 4, 8, 12 and 24 weeks after surgery. The repair tissues were sequentially analysed by arthroscopic surface imaging, OCT, and histology. The resulting images were compared to determine qualitative and quantitative features of surface roughness, repair tissue integration, and micro-architecture. Statistical analysis was performed using Student's t -testing and linear regression. RESULTS: OCT was able to identify the bone and cartilage interface in normal rabbit articular cartilage and regenerated cartilage at 24 weeks post chondrocyte implantation. OCT was able to identify hypertrophy at 4 and 8 weeks, and subtle surface fibrillations at 24 weeks, comparable with histological analysis at low magnification (20x). More importantly, OCT was able to detect embedded gaps between the repair tissue and surrounding host cartilage. CONCLUSION: Close correlation was observed between OCT and histological analysis of morphological features important to the assessment of articular cartilage repair. These results demonstrate that OCT is capable of providing immediate 'optical biopsy' of the rabbit articular cartilage repair tissue without damaging the specimen, and suggest that this new technique, if integrated with an arthroscope, can potentially be used in longitudinal studies of articular cartilage repair in vivo.     

Comparison of meshes, gels and ceramic for cartilage tissue engineering in vitro

Full-thickness articular cartilage defects lack the capacity of healing because of lack of blood supply and lack of chondrocyte proliferation around the injury site. These factors contribute to the difficulty of getting good healing of cartilage defects. Autologous chondrocyte transplantation has been proposed as a method for treating cartilage defects using chondrocytes grown in vitro, which are then transplanted into the defects using a periosteal flap to retain the cells at the defect site. Studies that followed have attempted to refine this technique by using a cell matrix to support the chondrocytes. The reason for adding a resorbable cell matrix support that acts as a temporary scaffold until the chondrocytes are capable of producing extracellular matrix. Moreover, such a matrix may help in maintaining chondrocyte differentiation and phenotype. In this study, we have investigated the biocompatibility between human chondrocytes and biomaterials that could be used as matrix implants. It is a comparative study in vitro that involves assessing the proliferation and differentiation of human articular chondrocytes cultured on different resorbable biomaterials. Human chondrocytes were isolated from collagenase digest of articular cartilage provided by patients undergoing total knee replacements for osteoarthritis from the non-involved areas of the knee. The chondrocytes were then allowed to proliferate in vitro to increase the number of cells available for study. After adequate multiplication, the cells were seeded onto different biomaterials and allowed to from a cell biomaterial construct. The biomaterials used in this study were collagen I, calcium alginate, agarose, polyglycolic acid and Bioglass 45S5. The cell–biomaterial constructs were then collected at specific time points 3, 7, 14 and 21 days for histological and biochemical studies. The assessment includes studying proliferation, differentiation and extracellular matrix production. This was performed by immunostaining for collagen I and II production and histochemistry staining for glycosaminoglycans. Chondrocyte proliferation was more effective on 3D gels compared to ceramics and mesh. Cells on Bioglass expressed the same collagen type and at the same proportion as that expressed by freshly isolated cells. Moreover, Bioglass has induced cells to re-differentiate after they lost their differentiation in monolayer culture. Overall, however, there was no clear relationship between the cell morphology and type of collagen produced. Bioactive glass seems to behave as a suitable material for chondrocyte tissue engineering because it can maintain a chondrocyte phenotype.     

Repair of rabbit articular surfaces with allograft chondrocytes embedded in collagen gel

In an attempt to repair articular cartilage, allograft articular chondrocytes embedded in collagen gel, were transplanted into full-thickness defects in rabbit articular cartilage. Twenty-four weeks after the transplantation, the defects were filled with hyaline cartilage, specifically synthesising Type II collagen. These chondrocytes were autoradiographically proven to have originated from the transplanted grafts. Assessed histologically the success rate was about 80%, a marked improvement over the results reported in previous studies on chondrocyte transplantation without collagen gel. By contrast, the defects without chondrocyte transplantation healed with fibrocartilage. Immunological enhancement induced by transplanted allogenic chondrocytes or collagen was not significant at eight weeks after treatment, so far as shown by both direct and indirect blastformation reactions. Thus, allogenic transplantation of isolated chondrocytes embedded in collagen gel appears to be one of the most promising methods for the restoration of articular cartilage.     

Value of autologous chondrocyte transplantation in experimental cartilage defects reconstruction. Part III--Microscopic analysis of reconstructed cartilage thickness and integration with surrounding tissue]

Lack of cartilage vascularization is the reason of its low regenerative potential. The aim of this part of the study was microscopic evaluation of repair tissue thickness and its integration with surrounding cartilage, after autologous chondrocyte transplantation. MATERIAL AND METHODS: Repair of partial thickness cartilage defect (ICRS III(o) grade) on distal femur joint surface was evaluated (25 adolescent rabbits). Procedures were performed in two groups: I--autologous chondrocyte transplantation under periosteal flap, II--periosteal graft. Chondrocytes were isolated from the cartilage specimens by enzymatic digestion and cultured in vitro. The follow-up periods were established at 4, 8, 12 weeks. Repair tissue was evaluated microscopically according to modified O'Driscoll scale. RESULTS: In group I, 8 weeks after the procedure most of defects were filled with the newly formed tissue almost completely. Regenerate thickness after 4 and 12 weeks usually exceeded 1/2 of surrounding cartilage. In group II, 8 weeks after the procedure regenerate thickness amounted to at least 1/2 of surrounding cartilage, but 4- and 12-week observation revealed the decreased repair tissue thickness. In group I, 4 weeks after the procedure regenerative tissue was well integrated with surrounding cartilage, and this trait still gradually increased with time. In group II, there was partial integration or no integration of repair tissue with surrounding cartilage. CONCLUSION: Obtained results indicate, that tissue formed after autologous chondrocyte transplantation with use of periosteal flap was better in its thickness and integration with surrounding cartilage, as compared to tissue formed after use of periosteum alone. Autologous chondrocyte transplantation can not guarantee complete filling of the cartilage defect with the graft tissue and full integration with surrounding cartilage, without three-dimensional scaffold application.