The Effect of Bone Marrow Stimulation for Cartilage Repair on the Subchondral Bone Plate.

PURPOSE: To investigate the effect of bone-marrow stimulation (BMS) on subchondral bone plate morphology and remodeling compared to untreated subchondral bone in a validated minipig model. METHODS: Three Göttingen minipigs received BMS with drilling as treatment for two chondral defects in each knee. The animals were euthanized after six months. Follow-up consisted of a histological semiquantitative evaluation using a novel subchondral bone scoring system and micro computed tomography (µCT) of the BMS subchondral bone. The histological and microstructural properties of the BMS-treated subchondral bone were compared to that of the adjacent healthy subchondral bone. RESULTS: The µCT analysis showed that subchondral bone treated with BMS had significantly higher connectivity density compared to adjacent untreated subchondral bone (26 1/mm3 vs. 21 1/mm3, P = 0.048). This was the only microstructural parameter showing a significant difference. The histological semiquantitative score differed significantly between the subchondral bone treated with BMS and the adjacent untreated subchondral (8.0 vs. 10 P = < 0.001). Surface irregularities were seen in 43% and bone overgrowth in 27% of the histological sections. Only sparse formation of bone cysts was detected (1%). CONCLUSIONS: BMS with drilling does not cause extensive changes to the subchondral bone microarchitecture. Furthermore, the morphology of BMS subchondral bone resembled that of untreated subchondral bone with almost no formation of bone cyst, but some surface irregularities and bone overgrowth.

No Effect of Platelet-Rich Plasma Injections as an Adjuvant to Autologous Cartilage Chips Implantation for the Treatment of Chondral Defects.

BACKGROUND: Repair of chondral injuries using cartilage chips has recently demonstrated clinical feasibility. Autologous platelet-rich plasma (PRP) is a potential promising technique for improving healing response during cartilage repair. PURPOSE: To assess the cartilage repair tissue quality after autologous cartilage chips treatment (CC) with and without repeated local injections of PRP for the treatment of full-thickness focal chondral defects of the knee. MATERIALS AND METHODS: Two full-thickness chondral defects (Ø = 6 mm) were created in the medial and lateral trochlea facets of each knee in 6 skeletally mature Göttingen minipigs. The 2 treatment groups were (1) CC with 1 weekly PRP injection for 3 weeks (n = 12) and (2) CC alone (n = 12). The animals were euthanized after 6 months. Samples of whole blood and PRP were analyzed for concentrations of platelets and nucleated cells. The composition of the cartilage repair tissue was assessed using gross appearance assessment, histomorphometry, and semiquantitative scoring (ICRS II). RESULTS: Histological evaluation demonstrated no significant difference in the content of hyaline cartilage (CC + PRP: 18.7% vs. CC: 19.6%), fibrocartilage (CC + PRP: 48.1% vs. CC: 51.8%), or fibrous tissue (CC + PRP: 22.7% vs. CC: 21.8%) between the treatment groups. Macroscopic evaluation did not demonstrate any difference between groups. CONCLUSIONS: PRP injections after CC in the treatment of full-thickness cartilage injuries demonstrated no beneficial effects in terms of macroscopic and histologic composition of cartilage repair tissue.

Particulated Cartilage for Chondral and Osteochondral Repair: A Review.

INTRODUCTION: Injuries to articular cartilage have a poor spontaneous repair potential and no gold standard treatment exist. Particulated cartilage, both auto- and allograft, is a promising new treatment method that circumvents the high cost of scaffold- and cell-based treatments. MATERIALS AND METHODS: A comprehensive database search on particulated cartilage was performed. RESULTS: Fourteen animal studies have found particulated cartilage to be an effective treatment for cartilage injuries. Many studies suggest that juvenile cartilage has increased regenerative potential compared to adult cartilage. Sixteen clinical studies on 4 different treatment methods have been published. (1) CAIS, particulated autologous cartilage in a scaffold, (2) Denovo NT, juvenile human allograft cartilage embedded in fibrin glue, (3) autologous cartilage chips-with and without concomitant bone grafting, and (4) augmented autologous cartilage chips. CONCLUSION: Implantation of allogeneic and autologous particulated cartilage provides a low cost and effective treatment alternative to microfracture and autologous chondrocyte implantation. The methods are promising, but large randomized controlled studies are needed.

No effect of platelet-rich plasma as adjuvant to bone marrow stimulation for the treatment of chondral defects in a large animal model.

BACKGROUND: Bone marrow stimulation (BMS) remains a dominant treatment strategy for symptomatic full thickness articular cartilage defects. Autologous platelet-rich plasma (PRP), may improve biological cartilage repair as an adjunct to BMS. OBJECTIVES: To assess the histological quality of cartilage repair after BMS with and without repeated local injections of PRP for the treatment of full-thickness focal chondral defects of the knee. METHODS: Two full-thickness chondral defects (Ø = 6 mm) were surgically performed in the medial and lateral trochlea of each knee in skeletally mature Göttingen minipigs. The two treatment groups with 12 defect for each groups were (1) BMS with one weekly PRP injection for 4 weeks, and (2) BMS alone. The animals were euthanized after 6 months. Samples of both whole blood and PRP were analysed with an automated hematology analyzer to determine the concentrations of platelets and nucleated cells. The composition of cartilage repair tissue was assessed using gross appearance assessment, histomorphometry and semi-quantitative scoring (ICRS II). RESULTS: The average fold increase in platelets was 10.2 ± 2.2. Leukocyte concentration increased in PRP samples by an average fold change of 7.2 ± 1.3. Our macroscopic findings showed that the defects in the BMS + PRP-treated group, were filled with an irregular, partially rough tissue similar to the BMS-treated group. No significant difference in amount of hyalin cartilage, fibrocartilage or fibrous tissue content and ICRS II scores was found between the groups. CONCLUSIONS: Four repeated local injections of leukocyte-rich PRP after BMS in the treatment of full-thickness cartilage injuries demonstrated no beneficial effects in terms of macroscopic and histological cartilage repair tissue quality.

Autologous Cartilage Chip Transplantation Improves Repair Tissue Composition Compared With Marrow Stimulation.

BACKGROUND: Repair of chondral injuries by use of cartilage chips has recently demonstrated clinical feasibility. PURPOSE: To investigate in vivo cartilage repair outcome of autologous cartilage chips compared with marrow stimulation in full-thickness cartilage defects in a minipig model. STUDY DESIGN: Controlled laboratory study. METHODS: Six Göttingen minipigs received two 6-mm chondral defects in the medial and lateral trochlea of each knee. The two treatment groups were (1) autologous cartilage chips embedded in fibrin glue (ACC) (n = 12) and (2) marrow stimulation (MST) (n = 12). The animals were euthanized after 6 months, and the composition of repair tissue was quantitatively determined using histomorphometry. Semiquantitative evaluation was performed by means of the International Cartilage Repair Society (ICRS) II score. Collagen type II staining was used to further evaluate the repair tissue composition. RESULTS: Significantly more hyaline cartilage was found in the ACC (17.1%) compared with MST (2.9%) group ( P < .01). Furthermore, the ACC group had significantly less fibrous tissue (23.8%) compared with the MST group (41.1%) ( P < .01). No significant difference in fibrocartilage content was found (54.7% for ACC vs 50.8% for MST). The ACC group had significantly higher ICRS II scores for tissue morphological characteristics, matrix staining, cell morphological characteristics, surface assessment, mid/deep assessment, and overall assessment ( P < .05). The ACC-treated defects had significantly more collagen type II staining (54.5%) compared with the MST-treated defects (28.1%) ( P < .05). CONCLUSION: ACC transplant resulted in improved quality of cartilage repair tissue compared with MST at 6 months postoperatively. CLINICAL RELEVANCE: Further studies are needed to investigate ACC as a possible alternative first-line treatment for focal cartilage injuries in the knee.

Autologous tissue transplantations for osteochondral repair.

Articular cartilage forms the articulating surface of synovial joints. Along with the synovial fluid it facilitates near frictionless movement in healthy joints. Injuries to articular cartilage in the knee are frequent and can lead to severe osteoarthritis, which is expected to affect 25% of the adult population by 2030. Hyaline cartilage does not regenerate spontaneously when injured and the current clinical treatment methods suffer from high cost and relatively high failure rates. This calls for new treatment methods to be developed. The backbone of developing new treatment methods for cartilage injuries is a reliable, cost-effective, clinically relevant large animal model. Study 1 aimed at developing such a model. We hypothesized that in the Göttingen minipig, the repair response of a selection of treatment methods would be similar to what is found in a clinical setting, and that two defects per knee, rather than one, could be applied without affecting the repair outcome. We found that the outcomes of the applied treatments were consistent with the outcomes in clinical studies. Furthermore, the use of two defects per knee did not have any significant effect on the repair response. The Göttingen minipig model was easy to handle, cost-effective, and provided a predictable repair response. Based on this study the use of two defects per knee in male Göttingen minipigs is recommended. The model has been implemented as the standard animal model for cartilage research at the Orthopedic Research Laboratory, Aarhus University Hospital. Synthetic osteochondral scaffolds represent an off-the-shelf, one-step treatment method, and preliminary clinical results have been promising. However, MRI investigations have shown issues related to subchondral healing. In study 2 we aimed at evaluating the osteochondral repair in 10 patients treated with the MaioRegen synthetic scaffold. Of the ten patients, two patients were re-operated due to treatment failure. CT imaging revealed that none of the eight remaining patients had complete regeneration of the subchondral bone. At 2.5 years, 6/8 patients had no or very limited (< 10%) bone formation in the defects and 2/8 had 50-75% bone formation in the treated defect. MRI showed no improvement at either one or 2.5 years compared with baseline. Clinical outcome scores were improved at 2.5 years. These results raise serious concerns about the biological repair potential of the MaioRegen scaffold. The use of the MaioRegen scaffold has been discontinued in Denmark as a result of this study. An alternative treatment approach for osteochondral lesions is combined transplantation of autologous bone graft and cartilage fragments, embedded in fibrin glue. In study 3 we investigated the early biological and clinical outcome of autologous dual-tissue transplantation (ADTT). ADTT is a combined autologous bone and cartilage chips transplantation for treatment of osteochondral injuries. It is easily applicable and bypasses the need for costly cell culturing or synthetic materials. After one year, all eight patients had significant improvements on MRI, CT and all clinical outcome scores. This study establishes ADTT as a promising, low-cost, treatment option for osteochondral injuries in the knee. To investigate the role of the implanted cartilage chips, we tested the isolated effect of the chips in the newly developed Göttingen minipig model. In study 4 we compared ADTT with autologous bone graft alone. The hypothesis was that the presence of cartilage chips would improve the quality of the repair tissue. Twelve Göttingen minipigs were included, and follow-up time was six and 12 months. Follow-up consisted of histomorphometry, immunohistochemistry, semi-quantitative scoring and CT. There was significantly more hyaline cartilage in the ADTT group compared with the autologous bone graft group at both six and 12 months. At both six and 12 months there were significantly more fibrocartilage in the ADTT group compared with the ABG group. The presence of cartilage chips in an osteochondral defect facilitated the formation of fibrocartilage as opposed to fibrous tissue at both six and 12 months. This study substantiates the chondrogenic role of cartilage chips in osteochondral defects, but questions the widely accepted repair mechanism involved in cartilage chip treatment methods. Further studies on the repair mechanism(s) involved are needed to improve the clinical application of autologous cartilage chips.

Collagen Type IV and Laminin Expressions during Cartilage Repair and in Late Clinically Failed Repair Tissues from Human Subjects.

OBJECTIVE: To identify the collagen type IV (Col4) isoform in articular cartilage and to evaluate the expressions of Col4 and laminin in the pericellular matrix (PCM) in damaged cartilage and during cartilage repair. DESIGN: The Col4 isoform was determined in chondrocytes isolated from 6 patients cultured up to 6 days and in 21% O2 or 1% O2, and the gene expression of Col4 α-chains was investigated. The distribution of Col4 and laminin in traumatically damaged cartilage (n = 7) and clinically failed cartilage repair (microfracture, TruFit, autologous chondrocyte implantation; n = 11) were investigated using immunohistochemistry. Normal human cartilage was used as control (n = 8). The distribution during clinical cartilage repair procedures was investigated in a minipig model with 6-month follow-up (untreated chondral, untreated osteochondral, microfracture, autologous chondrocyte implantation; n = 10). RESULTS: The Col4 isoform in articular cartilage was characterized as α1α1α2, which is an isoform containing antiangiogenic domains in the NC1-terminals (arresten and canstatin). In normal cartilage, laminin and Col4 was exclusively found in the PCM. High amounts (>50%) of Col4 in the PCM significantly decreased in damaged cartilage (P = 0.004) and clinically failed repair tissue (P < 0.001). Laminin was only found with high expression (>50%) in 4/8 of the normal samples, which was not statistically significantly different from damaged cartilage (P = 0.15) or failed cartilage repair (P = 0.054). CONCLUSIONS: Col4 in cartilage contain antiangiogenic domains and may play a role in the hypoxic environment in articular cartilage. Col4 and laminin was not found in the PCM of damaged and clinically failed repair.

Implantation of Autologous Cartilage Chips Improves Cartilage Repair Tissue Quality in Osteochondral Defects: A Study in Göttingen Minipigs.

BACKGROUND: Osteochondral injuries have poor endogenous healing potential, and no standard treatment has been established. The use of combined layered autologous bone and cartilage chips for treatment of osteochondral defects has shown promising short-term clinical results. PURPOSE/HYPOTHESIS: This study aimed to investigate the role of cartilage chips by comparing combined layered autologous bone and cartilage chips with autologous bone implantation alone in a Göttingen minipig model. The hypothesis was that the presence of cartilage chips would improve the quality of the repair tissue. STUDY DESIGN: Controlled laboratory study. METHODS: Twelve Göttingen minipigs received 2 osteochondral defects in each knee. The defects were randomized to autologous bone graft (ABG) combined with autologous cartilage chips (autologous dual-tissue transplantation [ADTT]) or ABG alone. Six animals were euthanized at 6 months and 6 animals were euthanized at 12 months. Follow-up evaluation consisted of histomorphometry, immunohistochemistry, semiquantitative scoring (International Cartilage Repair Society II), and computed tomography. RESULTS: There was significantly more hyaline cartilage in the ADTT group (25.8%) compared with the ABG group (12.8%) at 6 months after treatment. At 12 months, the fraction of hyaline cartilage in the ABG group had significantly decreased to 4.8%, whereas the fraction of hyaline cartilage in the ADTT group was unchanged (20.1%). At 6 and 12 months, there was significantly more fibrocartilage in the ADTT group (44% and 60.8%) compared with the ABG group (24.5% and 41%). The fraction of fibrous tissue was significantly lower in the ADTT group compared with the ABG group at both 6 and 12 months. The implanted cartilage chips stained >75% positive for collagen type 4 and laminin at both 6 and 12 months. Significant differences were found in a number of International Cartilage Repair Society II subcategories. The volume of the remaining bone defect significantly decreased from 6 to 12 months in both treatment groups; however, no difference in volume was found between the groups at either 6 or 12 months. CONCLUSION: The presence of cartilage chips in an osteochondral defect facilitated the formation of fibrocartilage as opposed to fibrous tissue at both 6 and 12 months posttreatment. The implanted chips were present in the defect and viable after 12 months. CLINICAL RELEVANCE: This study substantiates the chondrogenic role of cartilage chips in osteochondral defects.

Poor osteochondral repair by a biomimetic collagen scaffold: 1- to 3-year clinical and radiological follow-up.

INTRODUCTION: Treatment of osteochondral injuries is challenging, and no gold standard has been established. Layered cell-free scaffolds are a new treatment option for these defects. The aim of this study was to evaluate the osteochondral repair in patients treated with the MaioRegen(®) scaffold, a cell-free biomimetic scaffold consisting of type I collagen and hydroxyapatite. Treatment using this scaffold has previously shown promising clinical results. METHODS: Ten patients with osteochondral lesions in the knee (n = 6) or in the talus (n = 4) were enrolled. The patients underwent pre-operative MRI and CT scans and were assessed at 1- and 2.5-year timescales post-operatively. The cartilage and bone formations were evaluated semi-quantitatively using the MOCART score. Knee patients were clinically evaluated using KOOS, subjective IKDC and Tegner scores, whereas ankle patients were evaluated using AOFAS Hindfoot and Tegner scores. RESULTS: Two patients were re-operated and excluded from further follow-up due to treatment failure. None of the patients had complete regeneration of the subchondral bone evaluated using CT. At 2.5 years, 6/8 patients had no or very limited (<10 %) bone formation in the defects and 2/8 had 50-75 % bone formation in the treated defect. MRI showed no improvement in the MOCART score at any time point. The IKDC score improved from 41.3 to 80.7, and the KOOS pain subscale improved from 63.8 to 90.8 at 2.5-year follow-up. No improvement was found with the remaining KOOS subscales, the Tegner or AOFAS Ankle-Hindfoot score. CONCLUSION: Treatment of osteochondral defects in the ankle and knee joint with a biomimetic scaffold resulted in incomplete cartilage repair and poor subchondral bone repair at 1- and 2.5-year follow-up. Clinical significant improvements were observed. These results raise serious concerns about the biological repair potential of the MaioRegen(®) scaffold, and we advise to use the MaioRegen(®) scaffold with caution. LEVEL OF EVIDENCE: Prospective therapeutic study, Level IV.

Autologous Dual-Tissue Transplantation for Osteochondral Repair: Early Clinical and Radiological Results.

BACKGROUND: Numerous treatment methods for osteochondral repair have been implemented, including auto- and allogeneic osteochondral transplantations, combined bone and chondrocyte transplantations, and synthetic implants, but no gold standard treatment has been established. We present preliminary data on a combined autologous bone and cartilage chips: autologous dual-tissue transplantation (ADTT); an easily applicable, low-cost treatment option for osteochondral repair. The aim of this study was to investigate the early biological and clinical outcome of ADTT. MATERIALS: Eight patients (age 32 ± 7.5 years) suffering from osteochondritis dissecans (OCD) in the knee were enrolled. The OCD lesion was debrided and the osteochondral defect was filled with autologous bone, to a level at the base of the adjacent cartilage. Cartilage biopsies from the intercondylar notch were chipped and embedded within fibrin glue in the defect. Evaluation was performed using magnetic resonance imaging, computed tomography, and clinical scores, preoperative and 1 year postoperative. RESULTS: Cartilage tissue repair evaluated using MOCART score improved from 22.5 to 52.5 (P < 0.01). Computed tomography imaging demonstrated very good subchondral bone healing with all 8 patients having a bone filling of >80%. We found improvements 1 year postoperative in the International Knee Documentation Committee score (from 35.9 to 68.1, P < 0.01), Tegner score (from 2.6 to 4.7, P < 0.05), and Knee injury and Osteoarthritis Outcome Score pain, symptoms, sport/recreation and quality of life (P < 0.05). CONCLUSION: Treatment of OCD with ADTT resulted in very good subchondral bone restoration and good cartilage repair. Significant improvements in patient reported outcome was found at 1 year postoperative. This study suggests ADTT as a promising, low-cost, treatment option for osteochondral injuries.

Experimental articular cartilage repair in the Göttingen minipig: the influence of multiple defects per knee.

BACKGROUND: A gold standard treatment for articular cartilage injuries is yet to be found, and a cost-effective and predictable large animal model is needed to bridge the gap between in vitro studies and clinical studies. Ideally, the animal model should allow for testing of clinically relevant treatments and the biological response should be reproducible and comparable to humans. This allows for a reliable translation of results to clinical studies.This study aimed at verifying the Göttingen minipig as a pre-clinical model for articular cartilage repair by testing existing clinical cartilage repair techniques and evaluating the use of two defects per knee. METHODS: Sixteen fully mature Göttingen minipigs were used. The minipigs received bilateral trochlear osteochondral drill-hole defects or chondral defects (Ø 6 mm), either one defect per knee or two defects per knee. The defects were treated with one of the following: Matrix-induced autologous chondrocyte implantation (MACI), microfracture (MFx), autologous-dual-tissue transplantation (ADTT), autologous bone graft, autologous cartilage chips. Empty chondral and osteochondral defects were used as controls. MRI and CT were performed 3 and 6 month, histology was performed 6 month postoperative. RESULTS: The repair tissue varied in morphology from non-cartilaginous fibrous tissue to fibrocartilaginous tissue as seen on MRI, CT and histology at 6 month. The worst results were seen in the empty controls, while the best results were achieved with the MACI and ADTT treatment. The use of two defects per knee did not have any significant effect on the repair response. CONCLUSION: The outcomes of the applied treatments were consistent with the outcomes in clinical studies and it was possible to apply two defects per knee. The Göttingen minipig model was easy to handle, cost-effective and provided predictable outcome. Based on this study the use of two defects per knee, one in the medial and one in the lateral trochlear facet, in male Göttingen minipigs is recommended.

Increased chondrocyte seeding density has no positive effect on cartilage repair in an MPEG-PLGA scaffold.

PURPOSE: This study investigates the effect of cell seeding density on cartilage repair in matrix-assisted chondrocyte implantation in vitro and in vivo. METHODS: In vitro: Four different cell seeding densities of human chondrocytes were seeded onto a porous methoxy-polyethylene glycol-polylactic-co-glycolic acid scaffold (MPEG-PLGA) polymer scaffold ASEED™ (1.2 × 10(6), 4.0 × 10(6), 1.2 × 10(7) and 2.0 × 10(7) cells/cm(3)). The cartilage repair response was evaluated by relative gene expression of the chondrogenic markers sox9, collagen types I, II and X, and aggrecan, total DNA content and sulphated glycosaminoglycan synthesis. In vivo: Using a New Zealand white rabbit intercondylar osteochondral defect model, three different cell seeding densities (1.2 × 10(6), 4.0 × 10(6) and 1.2 × 10(7) cells/cm(3)) were tested with an empty scaffold as control. The cartilage repair response was evaluated using O'Driscoll score. RESULTS: In vitro: A significant difference (p < 0.05) in total DNA content was found at day 2 but not at day 7. The low cell seeding densities yielded the highest GAG content (p < 0.001) at day 7. Collagen type I was highest (p < 0.01) at the lowest density at day 7. In vivo: No significant difference was found between the 4 groups. CONCLUSIONS: No positive effect on cartilage repair was found using increased cell seeding density. LEVEL OF EVIDENCE: Controlled experimental study, Level II.

A novel nano-structured porous polycaprolactone scaffold improves hyaline cartilage repair in a rabbit model compared to a collagen type I/III scaffold: in vitro and in vivo studies.

PURPOSE: To develop a nano-structured porous polycaprolactone (NSP-PCL) scaffold and compare the articular cartilage repair potential with that of a commercially available collagen type I/III (Chondro-Gide) scaffold. METHODS: By combining rapid prototyping and thermally induced phase separation, the NSP-PCL scaffold was produced for matrix-assisted autologous chondrocyte implantation. Lyophilizing a water-dioxane-PCL solution created micro and nano-pores. In vitro: The scaffolds were seeded with rabbit chondrocytes and cultured in hypoxia for 6 days. qRT-PCR was performed using primers for sox9, aggrecan, collagen type 1 and 2. In vivo: 15 New Zealand White Rabbits received bilateral osteochondral defects in the femoral intercondylar grooves. Autologous chondrocytes were harvested 4 weeks prior to surgery. There were 3 treatment groups: (1) NSP-PCL scaffold without cells. (2) The Chondro-Gide scaffold with autologous chondrocytes and (3) NSP-PCL scaffold with autologous chondrocytes. Observation period was 13 weeks. Histological evaluation was made using the O'Driscoll score. RESULTS: In vitro: The expressions of sox9 and aggrecan were higher in the NSP-PCL scaffold, while expression of collagen 1 was lower compared to the Chondro-Gide scaffold. In vivo: Both NSP-PCL scaffolds with and without cells scored significantly higher than the Chondro-Gide scaffold when looking at the structural integrity and the surface regularity of the repair tissue. No differences were found between the NSP-PCL scaffold with and without cells. CONCLUSION: The NSP-PCL scaffold demonstrated higher in vitro expression of chondrogenic markers and had higher in vivo histological scores compared to the Chondro-Gide scaffold. The improved chondrocytic differentiation can potentially produce more hyaline cartilage during clinical cartilage repair. It appears to be a suitable cell-free implant for hyaline cartilage repair and could provide a less costly and more effective treatment option than the Chondro-Gide scaffold with cells.