Comparison Between Arthroscopic and Histological International Cartilage Repair Society Scoring Systems in Porcine Cartilage Repair Model.

OBJECTIVE: The arthroscopic and histological International Cartilage Repair Society (ICRS) scores are designed to evaluate cartilage repair quality. Arthroscopic ICRS score can give a maximum score of 12 and the histological score can give values between 0% and 100% for each of its 14 subscores. This study compares these methods in an animal cartilage repair model. This study hypothesizes that there is a significant correlation between these methods. DESIGN: A chondral defect was made in the medial femoral condyle of 18 pigs. Five weeks later, 9 pigs were treated with a novel recombinant human type III collagen/polylactide scaffold and 9 were left untreated to heal spontaneously. After 4 months, the medial condyles were evaluated with a simulated arthroscopy using the ICRS scoring system followed by a histological ICRS scoring. RESULTS: This porcine cartilage repair model produced repaired cartilage tissue ranging from good to poor repair tissue quality. The mean arthroscopic ICRS total score was 6.8 (SD = 2.2). Histological ICRS overall assessment subscore was 38.2 (SD = 31.1) and histological ICRS average points were 60.5 (SD = 19.5). Arthroscopic ICRS compared with histological ICRS average points or its overall assessment subscore showed moderate correlation (r = 0.49 and r = 0.50, respectively). The interrater reliability with the intraclass correlation coefficients for arthroscopic ICRS total scores, histological ICRS overall assessment subscore, and ICRS average points showed moderate to excellent reliability. CONCLUSIONS: Arthroscopic and histological ICRS scoring methods for repaired articular cartilage show a moderate correlation in the animal cartilage repair model.

Arthroscopic International Cartilage Repair Society Classification System Has Only Moderate Reliability in a Porcine Cartilage Repair Model.

BACKGROUND: The International Cartilage Repair Society (ICRS) score was designed for arthroscopic use to evaluate the quality of cartilage repair. PURPOSE: To evaluate the reliability of the ICRS scoring system using an animal cartilage repair model. STUDY DESIGN: Controlled laboratory study. METHODS: A chondral defect with an area of 1.5 cm2 was made in the medial femoral condyle of 18 domestic pigs. Five weeks later, 9 pigs were treated using a novel recombinant human type III collagen/polylactide scaffold, and 9 were left to heal spontaneously. After 4 months, the pigs were sacrificed, then 3 arthroscopic surgeons evaluated the medial femoral condyles via video-recorded simulated arthroscopy using the ICRS scoring system. The surgeons repeated the evaluation twice within a 9-month period using their recorded arthroscopy. RESULTS: The porcine cartilage repair model produced cartilage repair tissue of poor to good quality. The mean ICRS total scores for all observations were 6.6 (SD, 2.6) in arthroscopy, 5.9 (SD, 2.7) in the first reevaluation, and 6.2 (SD, 2.8) in the second reevaluation. The interrater reliability with the intraclass correlation coefficient (ICC) for the ICRS total scores (ICC, 0.46-0.60) and for each individual subscore (ICC, 0.26-0.71) showed poor to moderate reliability. The intrarater reliability with the ICC also showed poor to moderate reliability for ICRS total scores (ICC, 0.52-0.59) and for each individual subscore (ICC, 0.29-0.58). A modified Bland-Altman plot for the initial arthroscopy and for the 2 reevaluations showed an evident disagreement among the observers. CONCLUSION: In an animal cartilage repair model, the ICRS scoring system seems to have poor to moderate reliability. CLINICAL RELEVANCE: Arthroscopic assessment of cartilage repair using the ICRS scoring method has limited reliability. We need more objective methods with acceptable reliability to evaluate cartilage repair outcomes.

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment.

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.

Gas-foamed poly(lactide-co-glycolide) and poly(lactide-co-glycolide) with bioactive glass fibres demonstrate insufficient bone repair in lapine osteochondral defects.

Deep osteochondral defects may leave voids in the subchondral bone, increasing the risk of joint structure collapse. To ensure a stable foundation for the cartilage repair, bone grafts can be used for filling these defects. Poly(lactide-co-glycolide) (PLGA) is a biodegradable material that improves bone healing and supports bone matrix deposition. We compared the reparative capacity of two investigative macroporous PLGA-based biomaterials with two commercially available bone graft substitutes in the bony part of an intra-articular bone defect created in the lapine femur. New Zealand white rabbits (n = 40) were randomized into five groups. The defects, 4 mm in diameter and 8 mm deep, were filled with neat PLGA; a composite material combining PLGA and bioactive glass fibres (PLGA-BGf); commercial beta-tricalcium phosphate (ß-TCP) granules; or commercial bioactive glass (BG) granules. The fifth group was left untreated for spontaneous repair. After three months, the repair tissue was evaluated with X-ray microtomography and histology. Relative values comparing the operated knee with its contralateral control were calculated. The relative bone volume fraction (∆BV/TV) was largest in the ß-TCP group (p ≤ 0.012), which also showed the most abundant osteoid. BG resulted in improved bone formation, whereas defects in the PLGA-BGf group were filled with fibrous tissue. Repair with PLGA did not differ from spontaneous repair. The PLGA, PLGA-BGf, and spontaneous groups showed thicker and sparser trabeculae than the commercial controls. We conclude that bone repair with ß-TCP and BG granules was satisfactory, whereas the investigational PLGA-based materials were only as good as or worse than spontaneous repair.

Critical-sized cartilage defects in the equine carpus.

AIM: The horse joint, due to its similarity with the human joint, is the ultimate model for translational articular cartilage repair studies. This study was designed to determine the critical size of cartilage defects in the equine carpus and serve as a benchmark for the evaluation of new cartilage treatment options. MATERIAL AND METHODS: Circular full-thickness cartilage defects with a diameter of 2, 4, and 8 mm were created in the left middle carpal joint and similar osteochondral (3.5 mm in depth) defects in the right middle carpal joint of 5 horses. Spontaneously formed repair tissue was examined macroscopically, with MR and µCT imaging, polarized light microscopy, standard histology, and immunohistochemistry at 12 months. RESULTS: Filling of 2 mm chondral defects was good (77.8 ± 8.5%), but proteoglycan depletion was evident in Safranin-O staining and gadolinium-enhanced MRI (T1Gd). Larger chondral defects showed poor filling (50.6 ± 2.7% in 4 mm and 31.9 ± 7.3% in 8 mm defects). Lesion filling in 2, 4, and 8 mm osteochondral defects was 82.3 ± 3.0%, 68.0 ± 4.6% and 70.8 ± 15.4%, respectively. Type II collagen staining was seen in 9/15 osteochondral defects but only in 1/15 chondral defects. Subchondral bone pathologies were evident in 14/15 osteochondral samples but only in 5/15 chondral samples. Although osteochondral lesions showed better neotissue quality than chondral lesions, the overall repair was deemed unsatisfactory because of the subchondral bone pathologies. CONCLUSION: We recommend classifying 4 mm as critical osteochondral lesion size and 2 mm as critical chondral lesion size for cartilage repair research in the equine carpal joint model.

The Importance of Controlled Mismatch of Biomechanical Compliances of Implantable Scaffolds and Native Tissue for Articular Cartilage Regeneration.

Scaffolds for articular cartilage repair have to be optimally biodegradable with simultaneous promotion of hyaline cartilage formation under rather complex biomechanical and physiological conditions. It has been generally accepted that scaffold structure and composition would be the best when it mimics the structure of native cartilage. However, a reparative construct mimicking the mature native tissue in a healing tissue site presents a biological mismatch of reparative stimuli. In this work, we studied a new recombinant human type III collagen-polylactide (rhCol-PLA) scaffolds. The rhCol-PLA scaffolds were assessed for their relative performance in simulated synovial fluids of 1 and 4 mg/mL sodium hyaluronate with application of model-free analysis with Biomaterials Enhanced Simulation Test (BEST). Pure PLA scaffold was used as a control. The BEST results were compared to the results of a prior in vivo study with rhCol-PLA. Collectively the data indicated that a successful articular cartilage repair require lower stiffness of the scaffold compared to surrounding cartilage yet matching the strain compliance both in static and dynamic conditions. This ensures an optimal combination of load transfer and effective oscillatory nutrients supply to the cells. The results encourage further development of intelligent scaffold structures for optimal articular cartilage repair rather than simply trying to imitate the respective original tissue.

Recombinant human type II collagen hydrogel provides a xeno-free 3D micro-environment for chondrogenesis of human bone marrow-derived mesenchymal stromal cells.

Recombinant human type II collagen (rhCII) hydrogel was tested as a xeno-free micro-environment for the chondrogenesis of human bone marrow-derived mesenchymal stromal cells (BM-MSCs). The rhCII hydrogels were seeded with BM-MSCs and cultured in a xeno-free chondro-inductive medium for 14, 28 and 84 days. High-density pellet cultures served as controls. The samples were subjected to biochemical, histological and gene expression analyses. Although the cells deposited glycosaminoglycans into the extracellular space significantly more slowly in the rhCII hydrogels compared to the high-density pellets, a similar potential of matrix deposition was reached by the end of the 84-day culture. At day 28 of culture, the gene expression level for cartilage marker genes (i.e. genes encoding for Sox9 transcription factor, Collagen type II and Aggrecan) were considerably lower in the rhCII hydrogels than in the high-density pellets, but at the end of the 84-day culture period, all the cartilage marker genes analysed were expressed at a similar level. Interestingly, the expression of the matrix metallopeptidases (MMP)-13, MMP-14 and MMP-8, i.e. extracellular collagen network-degrading enzymes, were transiently upregulated in the rhCII hydrogel, indicating active matrix reorganization. This study demonstrated that the rhCII hydrogel functions as a xeno-free platform for BM-MSC chondrogenesis, although the process is delayed. The reversible catabolic reaction evoked by the rhCII hydrogel might be beneficial in graft integration in vivo and pinpoints the need to further explore the use of hydrogels containing recombinant extracellular matrix (ECM) proteins to induce the chondrogenesis of MSCs. Copyright © 2015 John Wiley & Sons, Ltd.

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.

Xeno-free chondrogenesis of bone marrow mesenchymal stromal cells: towards clinical-grade chondrocyte production.

Current cell-based cartilage therapies relay on articular cartilage-derived autologous chondrocytes as a cell source, which possesses disadvantages, such as, donor site morbidity and dedifferentiation of chondrocytes during in vitro expansion. Due to these and other limitations, novel cell sources and production strategies are needed. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a fascinating alternative, but they are not spontaneously capable of producing hyaline cartilage-like repair tissue in vivo. In vitro pre-differentiation of BM-MSCs could be used to produce chondrocytes for clinical applications. However, clinically compatible defined and xeno-free differentiation protocol is lacking. Hence, this study aimed to develop such chondrogenic differentiation medium for human BM-MSCs. We assessed the feasibility of the medium using three human BM-MSCs donors and validated the method by comparing BM-MSCs to three other cell types holding potential for articular cartilage repair. The effectiveness of the method was compared to conventional serum-free and commercially available chondrogenic differentiation media. The results show that the defined xeno-free differentiation medium is at least as efficient as conventionally used serum-free chondrogenic medium and performed significantly better on all cell types tested compared to the commercially available chondrogenic medium.

Optical projection tomography can be used to investigate spatial distribution of chondrocytes in three-dimensional biomaterial scaffolds for cartilage tissue engineering.

Biomaterial scaffolds have been used in autologous chondrocyte implantation to facilitate the repair of large lesions and to advance the formation of articular cartilage [Exp. Biol. Med. (Maywood) 237(1) (2012), 10-17]. Biomaterial scaffolds are usually three-dimensional (3-D) porous structures consisting of biodegradable materials to support articular cartilage formation. Adequate porosity of the scaffold is necessary for uniform cell distribution and cell attachment, and the density of the cells in the scaffold should be appropriate for cartilage formation [Cartilage 3(2) (2012), 108-117]. There have been only a restricted number of studies on the spatial distribution of cells in scaffolds, and on the role of this to cartilage formation [J. Biotechnol. 129 (2007), 516-531; Biotechnol. Progr. 14 (1998), 193-202; Biotechnol. Bioeng. 84 (2003), 205-214]. This may be due to the limited availability of appropriate visualization methods. Acquiring 3-D images throughout the scaffold by histology or confocal methods are not applicable to all types of scaffolds, and moreover, they are time consuming, laborious and thus not very feasible for a large scale analysis. To make the visualization of the spatial distribution of the cells easier in biomaterial scaffolds we have applied optical projection tomography (OPT). OPT microscope produces high-resolution 3-D images of both fluorescent and non-fluorescent specimens [Science 296(5567) (2002), 541-545]. Here we demonstrate that the OPT method can be used for the evaluation and visualization of the cell seeding method, spatial distribution and density of cells in biomaterial scaffolds and thus establish the OPT as a valid tool for analysis of cell distribution in cartilage tissue engineering samples.

An injectable, in situ forming type II collagen/hyaluronic acid hydrogel vehicle for chondrocyte delivery in cartilage tissue engineering.

In this study, chondrocytes were encapsulated into an injectable, in situ forming type II collagen/hyaluronic acid (HA) hydrogel cross-linked with poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4SPEG) and supplemented with the transforming growth factor ß1 (TGFß1). The chondrocyte-hydrogel constructs were cultured in vitro for 7 days and studied for cell viability and proliferation, morphology, glycosaminoglycan production, and gene expression. Type II collagen/HA/4SPEG formed a strong and stable hydrogel, and the chondrocytes remained viable during the encapsulation process and for the 7-day culture period. In addition, the encapsulated cells showed spherical morphology characteristic for chondrocytic phenotype. The cells were able to produce glycosaminoglycans into their extracellular matrix, and the gene expression of type II collagen and aggrecan, genes specific for differentiated chondrocytes, increased over time. The results indicate that the studied composite hydrogel with incorporated chondrogenic growth factor TGFß1 is able to maintain chondrocyte viability and characteristics, and thus, it can be regarded as potential injectable cell delivery vehicle for cartilage tissue engineering.

Affecting osteoblastic responses with in vivo engineered potato pectin fragments.

Pectins, complex plant-derived polysaccharides, are novel candidates for biomaterial nanocoatings. Pectic rhamnogalacturonan-I regions (RG-I) can be enzymatically treated to so-called modified hairy regions (MHR). We surveyed the growth and differentiation of murine preosteoblastic MC3T3-E1 cells on Petri dishes coated with RG-Is from native or genetically engineered potato tubers. Uncoated tissue culture polystyrene (TCPS) and aminated (AMI) dishes served as controls. MHRPTR_GAL sample was depleted of galactose (9 mol % galactose; 23 mol % arabinose) and MHRPTR_ARA of arabinose (61 mol % galactose; 6 mol % arabinose). Wild-type (modified hairy region from potato pectin (MHRP)_WT) fragment contained default amounts (58 mol % galactose; 13 mol % arabinose) of both sugars. Focal adhesions (FAs) indicating cellular attachment were quantified. Reverse transcriptase polymerase chain reaction (RT-PCR) of alkaline phosphatase and osteocalcin genes indicating osteoblastic differentiation was performed along with staining the produced calcium with tetracycline as an indicator of osteoblastic differentiation. Osteoblasts proliferated on all the samples to some extent. The control surfaces performed better than any of the pectin samples, of which the MHRP_WT seemed to function best. FA length was greater on MHRPTR_GAL than on other pectin samples, otherwise the mutants did not significantly deviate. RT-PCR results indicate that differences between the samples at the gene expression level might be even subtler. However, tetracycline-stained calcium-containing mineral was detected merely only on uncoated TCPS. These results indicate the possibility to affect bone cell growth with in vivo-modified pectin fragments, consecutively providing information on the significance of certain monosaccharides on the biocompatibility of these polysaccharides.

Dioxin-sensitive proteins in differentiating osteoblasts: effects on bone formation in vitro.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine-disrupting environmental pollutant which affects bone tissue, although the mechanistic basis of this action is far from clear. We adopted a proteome approach to investigate the disturbance of osteogenesis evoked by TCDD in an in vitro osteoblast differentiation model of rat mesenchymal stem cells. Eighteen individual proteins showed a significant change in abundance as results of ten days of TCDD exposure, at which time mRNA changes in osteoblast differentiation markers were also observed. These proteins were mostly involved in cytoskeleton organization and biogenesis, actin filament-based processes, protein transport, and folding. The alteration in cell architecture and increase in cell adhesion were confirmed by confocal microscopy. The TCDD-induced decrease in the expression of calcium-binding proteins may interfere with osteoblast calcium deposition, which was in fact reduced by TCDD. This is the first report investigating, at the protein expression level, the effect of TCDD during osteoblastic differentiation. Interestingly, MetaCore pathway analysis grouped the majority of these proteins around two principal nodes (c-fos and c-myc) suggesting that they may participate in the transcriptional activation of key pathways in TCDD-driven inhibition of osteoblast differentiation. Our findings provide evidence of new molecular players in the effects of TCDD on bone development.