Injectable Lyophilized Chitosan-Thrombin-Platelet-Rich Plasma (CS-FIIa-PRP) Implant to Promote Tissue Regeneration: In Vitro and Ex Vivo Solidification Properties.

Freeze-dried chitosan formulations solubilized in platelet-rich plasma (PRP) are currently evaluated as injectable implants with the potential for augmenting the standard of care for tissue repair in different orthopedic conditions. The present study aimed to shorten the solidification time of such implants, leading to an easier application and a facilitated solidification in a wet environment, which were direct demands from orthopedic surgeons. The addition of thrombin to the formulation before lyophilization was explored. The challenge was to find a formulation that coagulated fast enough to be applied in a wet environment but not too fast, which would make handling/injection difficult. Four thrombin concentrations were analyzed (0.0, 0.25, 0.5, and 1.0 NIH/mL) in vitro (using thromboelastography, rheology, indentation, syringe injectability, and thrombin activity tests) as well as ex vivo (by assessing the implant's adherence to tendon tissue in a wet environment). The biomaterial containing 0.5 NIH/mL of thrombin significantly increased the coagulation speed while being easy to handle up to 6 min after solubilization. Furthermore, the adherence of the biomaterial to tendon tissues was impacted by the biomaterial-tendon contact duration and increased faster when thrombin was present. These results suggest that our biomaterial has great potential for use in regenerative medicine applications.

Chitosan-platelet-rich plasma implants improve rotator cuff repair in a large animal model: Pilot study.

Freeze-dried formulations of chitosan can be solubilized in platelet-rich plasma (PRP) to form injectable implants that are used as an adjunct treatment during surgical repair of the rotator cuff. The purpose of the current study was to assess chitosan-PRP implant residency, test safety, and assess efficacy over standard-of-care controls in a sheep model of rotator cuff repair. The infraspinatus tendon was transected unilaterally and immediately repaired with suture anchors in 22 skeletally mature ewes. In treatment groups, formulations containing chitosan, trehalose, and calcium chloride were solubilized with autologous leukocyte-rich PRP and injected at the tendon-bone interface and on top of the repaired site (1 mL or 2 mL doses). Implant residency was assessed histologically at 1 day. Outcome measures included MRI assessment at baseline, 6 weeks, and 12 weeks, histopathology and clinical pathology. Chitosan-PRP implants were resident at the injection site at 1 day and induced recruitment of polymorphonuclear cells. The tendon gap, which corresponds to the length of abnormally hyperintense tissue attached to the humeral head, was decreased by treatment with the 2 mL dose when compared to controls at 12 weeks on MRI images. Some histological features were improved by the 2 mL dose treatment compared to controls at 12 weeks. There was no treatment-specific effect on all standard safety outcome measures, which suggests high safety. This study provides preliminary evidence on the safety and efficacy of chitosan-PRP implants in a large animal model that could potentially be translated to a clinical setting.

Chitosan-Platelet-Rich Plasma Implants Improve Rotator Cuff Repair in a Large Animal Model: Pivotal Study.

The purpose of this study was to assess the safety and efficacy of chitosan-platelet-rich plasma (PRP) hybrid implants used as an adjunct to surgical rotator cuff repair in a pivotal Good Laboratory Practice (GLP)-compliant study. The infraspinatus tendon was transected in 48 skeletally mature ewes and repaired with a transosseous-equivalent (TOE) technique. In the two treatment groups, a chitosan-PRP solution was injected at the footprint between the tendon and the bone and on top of the repaired site (2 mL or 3 mL doses, n = 12 per group). To further assess chitosan safety, a chitosan-water solution was injected at the same sites (3 mL, n = 12). Outcome measures included Magnetic Resonance Imaging (MRI) assessment and clinical pathology at 3 months and 6 months and histopathology at 6 months. The tendon gap was decreased at 3 months on MRI images and certain histopathological features were improved at 6 months by chitosan-PRP treatment compared to controls. The group treated with chitosan-water was not different from controls. Chitosan-PRP treatment induced no negative effects in the sheep, which suggests high safety. This study provides further evidence on the safety and efficacy of chitosan-PRP for rotator cuff repair augmentation, which could eventually be used in a clinical setting.

Robust Segmentation-Free Algorithm for Homogeneity Quantification in Images.

OBJECTIVE: Homogeneity is a notion used to describe images in various fields and is often linked to critical aspects of those fields. However, this term is rarely defined in the literature and no gold standard exists for its quantification. A few quantification algorithms have been proposed, but they lack both simplicity and robustness. As a result, the scientific community uses the notion of homogeneity in subjective analysis, preventing objective comparison of a large number of data or of different studies. The main objectives of this manuscript are to propose a definition of homogeneity and an algorithm for its quantification. METHOD: This algorithm, called MASQH, rely on a multi-scale, statistical and segmentation-free approach and outputs a single homogeneity index, which makes it robust and easy to use. RESULTS: The performance and reliability of the method are demonstrated through three case studies: Firstly, on synthetic images to study the behavior and assess the relevance of the algorithm in diverse situations and hence, in various potential fields. Secondly, on histological images derived from experimental chitosan-platelet-rich-plasma hybrid biomaterial, where the quantitative results are compared to a qualitative classification provided by an expert in the field. Thirdly, on experimental nanocomposites images for which results are compared to two other homogeneity quantification algorithms from the field of nanocomposites. CONCLUSION AND SIGNIFICANCE: By quantifying homogeneity, the MASQH method may help to compare disparate studies in the literature and quantitatively demonstrate the impact of homogeneity in various fields. The MASQH method is freely available online.

Quality of Cartilage Repair from Marrow Stimulation Correlates with Cell Number, Clonogenic, Chondrogenic, and Matrix Production Potential of Underlying Bone Marrow Stromal Cells in a Rabbit Model.

OBJECTIVE: Previous studies have shown that intrinsic behavior of subchondral bone marrow stem cells (BMSCs) is influenced by donors and locations. To understand the variability in cartilage repair outcomes following bone marrow stimulation, we tested the hypothesis that in vivo cartilage repair correlates with in vitro biological properties of BMSCs using a rabbit model. METHODS: Full-thickness cartilage defects were created in the trochlea and condyle in one knee of skeletally mature New Zealand White rabbits (n = 8) followed by microdrilling. Three-week repair tissues were analyzed by macroscopic International Cartilage Repair Society (ICRS) scores, O'Driscoll histological scores, and Safranin-O (Saf-O) and type-II collagen (Coll-II) % stain. BMSCs isolated from contralateral knees were assessed for cell yield, surface marker expression, CFU-f, %Saf-O, and %Coll-II in pellet culture followed by correlation analyses with the above cartilage repair responses. RESULTS: In vivo cartilage repair scores showed strong, positive correlation with cell number, clonogenic, chondrogenic, and matrix production (Coll-II, GAG) potential of in vitro TGF-ßIII stimulated BMSC cultures. Trochlear repair showed clear evidence of donor dependency and strong correlation was observed for interdonor variation in repair and the above in vitro properties of trochlear BMSCs. Correlation analyses indicated that donor- and location-dependent variability observed in cartilage repair can be attributed to variation in the properties of BMSCs in underlying subchondral bone. CONCLUSION: Variation in cell number, clonogenic, chondrogenic, and matrix production potential of BMSCs correlated with repair response observed in vivo and appear to be responsible for interanimal variability as well as location-dependent repair.

A novel image analysis algorithm reveals that media conditioned with chitosan and platelet-rich plasma biomaterial dose dependently increases fibroblast migration in a scratch assay.

Chitosan (CS) and Platelet-Rich Plasma (PRP) both display interesting properties for wound healing applications. A hybrid CS-PRP biomaterial was previously developped, consisting of a freeze dried CS formulation solubilized in PRP that promotes tissue repair and regeneration. The purpose of the current study was to investigate the ability of the CS-PRP biomaterial to stimulate cell migrationin vitro. Scratch assays revealed that CS-PRP significantly stimulates the migration rate of cells compared to cells in culture medium but not differently than PRP alone. The increase in the migration rate is dose-dependent at low dose and reaches a plateau corresponding with maximum cell motility. Cell migration rate as a function of the number of platelets that have degranulated in culture medium (to which total concentration of growth factors contributing to cell response is proportionnal), follows a modified Hill model. To analyze photographs taken during the assay and follow cell migration, an open source image analysis algorithm was developed: SAMScratch (Systematic Area Measurement of Scratch - available here:https://github.com/Biomaterials-and-Cartilage-Laboratory/SAM-Scratch). Compared with other existing analysis tools, the algorithm is precise in the determination of the scratch area and performs equally well with usual and challenging images. This study resulted in the creation of a freely available application for scratch assay analysis and provided evidence that CS-PRP implants hold promise for treatment of wounds.

Multiple platelet-rich plasma preparations can solubilize freeze-dried chitosan formulations to form injectable implants for orthopedic indications.

BACKGROUND: Platelet-rich plasma (PRP) has been used to solubilize freeze-dried chitosan (CS) formulations to form injectable implants for tissue repair. OBJECTIVE: To determine whether the in vitro performance of the formulations depends on the type of PRP preparation used to solubilize CS. METHODS: Formulations containing 1% (w/v) CS with varying degrees of deacetylation (DDA 80.5-84.8%) and number average molar mass (Mn 32-55 kDa), 1% (w/v) trehalose and 42.2 mM calcium chloride were freeze-dried. Seven different PRP preparations were used to solubilize the formulations. Controls were recalcified PRP. RESULTS: CS solubilization was achieved with all PRP preparations. CS-PRP formulations were less runny than their corresponding PRP controls. All CS-PRP formulations had a clotting time below 9 minutes, assessed by thromboelastography, while the leukocyte-rich PRP controls took longer to coagulate (>32 min), and the leukocyte-reduced PRP controls did not coagulate in this dynamic assay. In glass culture tubes, all PRP controls clotted, expressed serum and retracted (43-82% clot mass lost) significantly more than CS-PRP clots (no mass lost). CS dispersion was homogenous within CS-PRP clots. CONCLUSIONS: In vitro performance of the CS-PRP formulations was comparable for all types of PRPs assessed.

Injectable freeze-dried chitosan-platelet-rich-plasma implants improve marrow-stimulated cartilage repair in a chronic-defect rabbit model.

Bone-marrow stimulation (BMS) improves knee-joint function but elicits incomplete repair. Liquid chitosan (CS)-glycerol phosphate/blood clots have been shown to improve BMS-based cartilage repair. Platelet-rich-plasma (PRP)-a rich source of growth factors and cytokines-improves recruitment and chondrogenic potential of subchondral mesenchymal stem cells. We hypothesised that repair response in a rabbit chronic-defect model will improve when freeze-dried CS/PRP is used to augment BMS. Bilateral trochlear defects created in New Zealand white rabbits were allowed to progress to a chronic stage over 4 weeks. Chronic defects were debrided and treated by BMS in second surgery, then augmented with PRP (BMS + PRP) or freeze-dried CS/PRP implants (BMS + CS/PRP). The quality of 8-week repair tissue was assessed by macroscopic, histological, and micro computed tomography (Micro-CT) analysis. ICRS macroscopic scores indicated fibrocartilaginous or fibrous repair in control defects that were improved in the BMS + CS/PRP group. An overall improvement in repair in BMS + CS/PRP group was further confirmed by higher O'Driscoll scores, %Saf-O and %Coll-II values. Micro-CT analysis of subchondral bone indicated ongoing remodelling with repair still underway. Quality and quantity of cartilage repair was improved when freeze-dried CS/PRP implants were used to augment BMS in a chronic defect model.

Freeze-dried chitosan-platelet-rich plasma implants improve supraspinatus tendon attachment in a transosseous rotator cuff repair model in the rabbit.

Rotator cuff tears result in shoulder pain, stiffness, weakness and loss of motion. After surgical repair, high failure rates have been reported based on objective imaging and it is recognized that current surgical treatments need improvement. The aim of the study was to assess whether implants composed of freeze-dried chitosan (CS) solubilized in autologous platelet-rich plasma (PRP) can improve rotator cuff repair in a rabbit model. Complete tears were created bilaterally in the supraspinatus tendon of New Zealand White rabbits ( n = 4 in a pilot feasibility study followed by n = 13 in a larger efficacy study), which were repaired using transosseous suturing. On the treated side, CS-PRP implants were injected into the transosseous tunnels and the tendon itself, and healing was assessed histologically at time points ranging from one day to two months post-surgery. CS-PRP implants were resident within transosseous tunnels and adhered to tendon surfaces at one day post-surgery and induced recruitment of polymorphonuclear cells from 1 to 14 days. CS-PRP implants improved attachment of the supraspinatus tendon to the humeral head through increased bone remodelling at the greater tuberosity and also inhibited heterotopic ossification of the supraspinatus tendon at two months. In addition, the implants did not induce any detectable deleterious effects. This preliminary study provides the first evidence that CS-PRP implants could be effective in improving rotator cuff tendon attachment in a small animal model.

Freeze-Dried Chitosan-Platelet-Rich Plasma Implants for Rotator Cuff Tear Repair: Pilot Ovine Studies.

Rotator cuff tears are a very common shoulder pathology. Different suturing techniques have been used for surgical cuff repair, but failure of healing remains a significant clinical challenge. The objective of this study was to establish and compare chronic and acute ovine rotator cuff tear models in our laboratory and investigate the feasibility of using chitosan (CS)-platelet-rich plasma (PRP) implants in conjunction with suture anchors to treat rotator cuff tears in large animal models. Repair with suture anchors only was used as control. In two preliminary pilot studies, unilateral full-thickness tears were created in the infraspinatus (ISP) tendon of mature female Texel-cross sheep. In the chronic model (n = 4 sheep), the tendons were capped with silicon and allowed to retract for 6 weeks, leading to degenerative changes, whereas the tendons were immediately repaired in the acute model (n = 4 sheep). Transected ISP tendons were reattached with suture anchors and, in the case of treated shoulders, implants composed of freeze-dried CS solubilized in autologous PRP were additionally applied to the tendon-bone interface and on top of the repaired site. The chronic defect model induced significant tendon degeneration and retraction, which made repair more challenging than in the acute defect model. Half the tendons in the chronic repair model were found to be irrepairable at 6 weeks. In the other half, the tendons could not be reattached to the footprint due to significant retraction, which made this a model of tissue formation in a gap. In contrast, the acute tendon repair model was executed easily. Extensive bone remodeling and tissue ingrowth at the tendon-bone interface were observed in the case of treatment with anchors + CS-PRP in both models, suggesting that CS-PRP implants could potentially modulate rotator cuff healing processes in large animal models.

Augmentation Techniques for Meniscus Repair.

Menisci display exquisitely complex structure and play an essential weight-bearing role in the knee joint. A torn meniscus is one of the most common knee injuries which can result in pain and mechanical abnormalities. Tear location is one aspect which determines the endogenous healing response; tears that occur in the peripheral densely vascularized zone of the meniscus have the potential to heal while the healing capacity is more limited in the less vascularized inner zones. Meniscectomy was once widely performed, but led to poor radiographic and patient-reported mid- and long-term outcomes. After the advent of arthroscopy, orthopaedic opinion in the 1980s has been swaying toward salvaging or repairing the torn meniscus tissue to prevent osteoarthritis rather than performing meniscectomy. Meniscus repair in young active individuals has been shown to be effective, reproducible, and reliable if indications are met; however, only a small proportion of all tears are considered repairable with available technologies. Biological augmentation techniques and meniscus tissue engineering strategies are being devised to enhance the likelihood and rate of healing in meniscus repair. Preclinical and clinical studies have shown that introduction of cellular elements of the blood, bone marrow, and related growth factors have the potential to enhance meniscus repair. This article reviews the current state of clinical management of meniscus tears (primary repair) as well as augmentation techniques to improve healing by meniscus wrapping with extracellular matrix materials, trephination, synovial rasping and abrasion, fibrin/blood clot placement, and platelet-rich plasma injections. In addition, the rationale for using polymer/autologous blood component implants to improve meniscus repair will be discussed.

Bone Marrow Progenitor Cells Isolated from Young Rabbit Trochlea Are More Numerous and Exhibit Greater Clonogenic, Chondrogenic, and Osteogenic Potential than Cells Isolated from Condyles.

OBJECTIVE: Bone marrow stimulation procedures initiate repair by fracturing or drilling subchondral bone at base of cartilaginous defect. Earlier studies have shown that defect location and animal age affect cartilage repair outcome, suggesting a strong influence of structural and biological characteristics of subchondral bone. Here, we analyzed comprehensive biological characteristics of bone marrow progenitor cells (BMPCs) in subchondral bone of young and old rabbit condyle and trochlea. We tested the hypothesis that in vitro biological properties of BMPCs are influenced by location, age of donor and method of their isolation. DESIGN: In vitro biological properties, including cell yield, colony-forming unit fibroblasts (CFU-f), surface marker expression, and differentiation potential were determined. Comparisons were carried out between trochlea versus condyle and epiphyseal versus metaphyseal bone using old ( N = 5) and young animal knees ( N = 8) to generate collagenase and explant-derived BMPC cultures. RESULTS: CFU-f, cell yield, expression of stem cell markers, and osteogenic differentiation were significantly superior for younger animals. Trochlear subchondral bone yielded the most progenitors with the highest clonogenic potential and cartilaginous matrix expression. Trochlear collagenase-derived BMPCs had higher clonogenic capacity than explant-derived ones. Epiphyseal cells generated a larger chondrogenic pellet mass than metaphyseal-derived BMPCs. All older pellet cultures and one non-responder young rabbit failed to accumulate glycosaminoglycans (GAGs). CONCLUSION: Taken together, these results suggest that properties intrinsic to subchondral progenitors could significantly influence cartilage repair potential, and could partly explain variability in cartilage repair outcomes using same cartilage repair approach.

siRNA Delivery with Chitosan: Influence of Chitosan Molecular Weight, Degree of Deacetylation, and Amine to Phosphate Ratio on in Vitro Silencing Efficiency, Hemocompatibility, Biodistribution, and in Vivo Efficacy.

Chitosan (CS) shows in vitro and in vivo efficacy for siRNA delivery but with contradictory findings for incompletely characterized systems. For understanding which parameters produce effective delivery, a library of precisely characterized chitosans was produced at different degrees of deacetylation (DDAs) and average molecular weights (Mn). Encapsulation and transfection efficiencies were characterized in vitro. Formulations were selected to examine the influence of Mn and N:P ratio on nanoparticle uptake, metabolic activity, genotoxicity, and in vitro transfection. Hemocompatibility and in vivo biodistribution were then investigated for different Mn, N:P ratios, and doses. Nanoparticle uptake and gene silencing correlated with increased surface charge, which was obtained at high DDA and high Mn. A minimum polymer length of ∼60-70 monomers (∼10 kDa) was required for stability and knockdown. In vitro knockdown was equivalent to lipid control with no metabolic or genotoxicity. An inhibitory effect of serum on biological performance was dependent on DDA, Mn, and N:P. In vivo biodistribution in mice show accumulation of nanoparticles in kidney with 40-50% functional knockdown.

Lyophilisation and concentration of chitosan/siRNA polyplexes: Influence of buffer composition, oligonucleotide sequence, and hyaluronic acid coating.

Chitosan (CS)/siRNA polyplexes have great therapeutic potential for treating multiple diseases by gene silencing. However, clinical application of this technology requires the development of concentrated, hemocompatible, pH neutral formulations for safe and efficient administration. In this study we evaluate physicochemical properties of chitosan polyplexes in various buffers at increasing ionic strengths, to identify conditions for freeze-drying and rehydration at higher doses of uncoated or hyaluronic acid (HA)-coated polyplexes while maintaining physiological compatibility. Optimized formulations are used to evaluate the impact of the siRNA/oligonucleotide sequence on polyplex physicochemical properties, and to measure their in vitro silencing efficiency, cytotoxicity, and hemocompatibility. Specific oligonucleotide sequences influence polyplex physical properties at low N:P ratios, as well as their stability during freeze-drying. Nanoparticles display greater stability for oligodeoxynucleotides ODN vs siRNA; AT-rich vs GC-rich; and overhangs vs blunt ends. Using this knowledge, various CS/siRNA polyplexes are prepared with and without HA coating, freeze-dried and rehydrated at increased concentrations using reduced rehydration volumes. These polyplexes are non-cytotoxic and preserve silencing activity even after rehydration to 20-fold their initial concentration, while HA-coated polyplexes at pH∼7 also displayed increased hemocompatibility. These concentrated formulations represent a critical step towards clinical development of chitosan-based oligonucleotide intravenous delivery systems.

Chitosan inhibits platelet-mediated clot retraction, increases platelet-derived growth factor release, and increases residence time and bioactivity of platelet-rich plasma in vivo.

Platelet-rich plasma (PRP) has been used to treat different orthopedic conditions, however, the clinical benefits of using PRP remain uncertain. Chitosan (CS)-PRP implants have been shown to improve meniscus, rotator cuff and cartilage repair in pre-clinical models. The purpose of this current study was to investigate in vitro and in vivo mechanisms of action of CS-PRP implants. Freeze-dried formulations containing 1% (w/v) CS (80% degree of deacetylation and number average molar mass 38 kDa), 1% (w/v) trehalose as a lyoprotectant and 42.2 mM calcium chloride as a clot activator were solubilized in PRP. Gravimetric measurements and molecular/cellular imaging studies revealed that clot retraction is inhibited in CS-PRP hybrid clots through physical coating of platelets, blood cells and fibrin strands by chitosan, which interferes with platelet aggregation and platelet-mediated clot retraction. Flow cytometry and ELISA assays revealed that platelets are activated and granules secreted in CS-PRP hybrid clots and that cumulative release of platelet-derived growth factor (PDGF-AB) and epidermal growth factor is higher from CS-PRP hybrid clots compared to PRP clots in vitro. Finally, CS-PRP implants resided for up to 6 weeks in a subcutaneous implantation model and induced cell recruitment and granulation tissue synthesis, confirming greater residency and bioactivity compared to PRP in vivo.

Electromechanical probe and automated indentation maps are sensitive techniques in assessing early degenerated human articular cartilage.

Recent advances in the development of new drugs to halt or even reverse the progression of Osteoarthritis at an early-stage requires new tools to detect early degeneration of articular cartilage. We investigated the ability of an electromechanical probe and an automated indentation technique to characterize entire human articular surfaces for rapid non-destructive discrimination between early degenerated and healthy articular cartilage. Human cadaveric asymptomatic articular surfaces (four pairs of distal femurs and four pairs of tibial plateaus) were used. They were assessed ex vivo: macroscopically, electromechanically, (maps of the electromechanical quantitative parameter, QP, reflecting streaming potentials), mechanically (maps of the instantaneous modulus, IM), and through cartilage thickness. Osteochondral cores were also harvested from healthy and degenerated regions for histological assessment, biochemical analyses, and unconfined compression tests. The macroscopic visual assessment delimited three distinct regions on each articular surface: Region I was macroscopically degenerated, region II was macroscopically normal but adjacent to regions I and III was the remaining normal articular surface. Thus, each extracted core was assigned to one of the three regions. A mixed effect model revealed that only the QP (p < 0.0001) and IM (p < 0.0001) were able to statistically discriminate the three regions. Effect size was higher for QP and IM than other assessments, indicating greater sensitivity to distinguish early degeneration of cartilage. When considering the mapping feature of the QP and IM techniques, it also revealed bilateral symmetry in a moderately similar distribution pattern between bilateral joints. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:858-867, 2017.

Rotator cuff repair: a review of surgical techniques, animal models, and new technologies under development.

Rotator cuff tears are the most common musculoskeletal injury occurring in the shoulder. Current surgical repair fails to heal in 20% to 95% of patients, depending on age, size of the tear, smoking, time of repair, tendon quality, muscle quality, healing response, and surgical treatments. These problems are worsened by the limited healing potential of injured tendons attributed to the presence of degenerative changes and relatively poor vascularity of the cuff tendons. Development of new techniques to treat rotator cuff tears requires testing in animal models to assess safety and efficacy before clinical testing. Hence, it is important to evaluate appropriate animal models for rotator cuff research with degeneration of tendons, muscular atrophy, and fatty infiltration similar to humans. This report reviews current clinical treatments and preclinical approaches for rotator cuff tear repair. The review will focus on current clinical surgical treatments, new repair strategies under clinical and preclinical development, and will also describe different animal models available for rotator cuff research. These findings and future directions for rotator cuff tear repair will be discussed.

Chondroinduction Is the Main Cartilage Repair Response to Microfracture and Microfracture With BST-CarGel: Results as Shown by ICRS-II Histological Scoring and a Novel Zonal Collagen Type Scoring Method of Human Clinical Biopsy Specimens.

BACKGROUND: Current cartilage repair histological scoring systems are unable to explain the relationship between collagen type II deposition and overall repair quality. PURPOSE/HYPOTHESIS: The purpose of this study was to develop a novel zonal collagen type (ZCT) 5-point scoring system to measure chondroinduction in human clinical biopsy specimens collected after marrow stimulation. The hypothesis was that the ZCT scores would correlate with the International Cartilage Repair Society-II (ICRS-II) overall histological repair assessment score and glycosaminoglycan (GAG) content. STUDY DESIGN: Descriptive laboratory study. METHODS: After optimizing safranin O staining for GAG and immunostaining for human collagen type II and type I (Col2 and Col1, respectively), serial sections from clinical osteochondral repair biopsy specimens (13 months after microfracture or microfracture with BST-CarGel; n = 39 patients) were stained and 3 blinded readers performed histomorphometry for percentage of staining, ICRS-II histological scoring, polarized light microscopy (PLM) scoring, and 5-point ZCT scoring based on tidemark morphology, zonal distribution of Col2 and Col1, and Col1 percentage stain. Because 1 biopsy specimen was missing bone, 38 biopsy specimens were evaluated for ICRS-II, PLM, and ZCT scores. RESULTS: Chondroinduction was identified in 21 biopsy specimens as a Col2 matrix fused to bone that spanned the deep-middle-superficial zones ("full-thickness hyaline repair"), deep-middle zones, or deep zone ("stalled hyaline") that was covered with a variable-thickness Col1-positive matrix, and was scored, respectively, as ZCT = 1 (n = 4 biopsy specimens), ZCT = 2 (n = 6) and ZCT = 3 (n = 11). Other biopsy specimens (n = 17) were fibrocartilage (n = 9; ZCT = 4), fibrous tissue (n = 4, ZCT = 5), or non-marrow derived (n = 4; ZCT = 0). Non-marrow derived tissue had a mean mature tidemark score of 84 out of 100 versus a regenerating tidemark score of 24 for all other biopsy specimens (P = .005). Both "stalled hyaline" repair and fibrocartilage had the same mean Col2 percentage stain; however, fibrocartilage was distinguished by heavy Col1 deposits in the deep zone, a 2-fold higher mean Col1 percentage stain (P = .001), and lower surface integrity (P = .03). ZCT scores correlated with GAG content and the ICRS-II overall assessment score, especially when combined with the PLM score for collagen organization (R = 0.82). Histological scores of the deep zone strongly predicted the ICRS-II overall assessment score (R = 0.99). CONCLUSION: The ICRS-II overall repair assessment score and GAG content correlated with the extent of Col2 deposition free of fibrosis in the deep/middle zone rather than bulk accumulation of Col2. CLINICAL RELEVANCE: Biopsy tissue from the BST-CarGel randomized clinical trial (microfracture without and with BST-CarGel, as treatment groups were not unblinded) showed regenerated tissue consistent with a chondroinduction mechanism in at least half of the treated lesions.

Interspecies comparison of subchondral bone properties important for cartilage repair.

Microfracture repair tissue in young adult humans and in rabbit trochlea is frequently of higher quality than in corresponding ovine or horse models or in the rabbit medial femoral condyle (MFC). This may be related to differences in subchondral properties since repair is initiated from the bone. We tested the hypothesis that subchondral bone from rabbit trochlea and the human MFC are structurally similar. Trochlea and MFC samples from rabbit, sheep, and horse were micro-CT scanned and histoprocessed. Samples were also collected from normal and lesional areas of human MFC. The subchondral bone of the rabbit trochlea was the most similar to human MFC, where both had a relatively thin bone plate and a more porous and less dense character of subchondral bone. MFC from animals all displayed thicker bone plates, denser and less porous bone and thicker trabeculae, which may be more representative of older or osteoarthritic patients, while both sheep trochlear ridges and the horse lateral trochlea shared some structural features with human MFC. Since several cartilage repair procedures rely on subchondral bone for repair, subchondral properties should be accounted for when choosing animal models to study and test procedures that are intended for human cartilage repair.

Bone marrow stimulation of the medial femoral condyle produces inferior cartilage and bone repair compared to the trochlea in a rabbit surgical model.

The influence of the location of cartilage lesions on cartilage repair outcome is incompletely understood. This study compared cartilage and bone repair in medial femoral condylar (MFC) versus femoral trochlear (TR) defects 3 months after bone marrow stimulation in mature rabbits. Intact femurs from adult rabbits served as controls. Results from quantitative histomorphometry and histological scoring showed that bone marrow stimulation produced inferior soft tissue repair in MFC versus TR defects, as indicated by significantly lower % Fill (p = 0.03), a significant increase in collagen type I immunostaining (p < 0.00001) and lower O'Driscoll scores (p < 0.05). 3D micro-CT analysis showed that repaired TR defects regained normal un-operated values of bone volume fraction, trabecular thickness, and trabecular number, whereas in MFC defects the repaired bone architecture appeared immature and less dense compared to intact un-operated MFC controls (p < 0.0001). Severe medial meniscal damage was found in 28% of operated animals and was strongly correlated with (i) low cartilage defect fill, (ii) incomplete bone repair in MFC, and (iii) with a more posterior defect placement in the weight-bearing region. We conclude that the location of cartilage lesions influences cartilage repair, with better outcome in TR versus MFC defects in rabbits. Meniscal degeneration is associated with cartilage damage.