Caroline D. Hoemann

Novel injectable neutral solutions of chitosan form biodegradable gels in situ

A novel approach to provide, thermally sensitive neutral solutions based on chitosan/polyol salt combinations is described. These formulations possess a physiological pH and can be held liquid below room temperature for encapsulating living cells and therapeutic proteins; they form monolithic gels at body temperature. When injected in vivo the liquid formulations turn into gel implants in situ. This system was used successfully to deliver biologically active growth factors in vivo as well as an encapsulating matrix for living chondrocytes for tissue engineering applications. This study reports for the first time the use of polymer/polyol salt aqueous solutions as gelling systems, suggesting the discovery of a prototype for a new family of thermosetting gels highly compatible with biological compounds.

Chitosan-Glycerol Phosphate/Blood Implants Improve Hyaline Cartilage Repair in Ovine Microfracture Defects

BACKGROUND Microfracture is a surgical procedure that is used to treat focal articular cartilage defects. Although joint function improves following microfracture, the procedure elicits incomplete repair. As blood clot formation in the microfracture defect is an essential initiating event in microfracture therapy, we hypothesized that the repair would be improved if the microfracture defect were filled with a blood clot that was stabilized by the incorporation of a thrombogenic and adhesive polymer, specifically, chitosan. The objectives of the present study were to evaluate (1) blood clot adhesion in fresh microfracture defects and (2) the quality of the repair, at six months postoperatively, of microfracture defects that had been treated with or without chitosan-glycerol phosphate/blood clot implants, using a sheep model. METHODS In eighteen sheep, two 1-cm2 full-thickness chondral defects were created in the distal part of the femur and treated with microfracture; one defect was made in the medial femoral condyle, and the other defect was made in the trochlea. In four sheep, microfracture defects were created bilaterally; the microfracture defects in one knee received no further treatment, and the microfracture defects in the contralateral knee were filled with chitosan-glycerol phosphate/autologous whole blood and the implants were allowed to solidify. Fresh defects in these four sheep were collected at one hour postoperatively to compare the retention of the chitosan-glycerol phosphate/blood clot with that of the normal clot and to define the histologic characteristics of these fresh defects. In the other fourteen sheep, microfracture defects were made in only one knee and either were left untreated (control group; six sheep) or were treated with chitosan-glycerol phosphate/blood implant (treatment group; eight sheep), and the quality of repair was assessed histologically, histomorphometrically, and biochemically at six months postoperatively. RESULTS In the defects that were examined one hour postoperatively, chitosan-glycerol phosphate/blood clots showed increased adhesion to the walls of the defects as compared with the blood clots in the untreated microfracture defects. After histological processing, all blood clots in the control microfracture defects had been lost, whereas chitosanglycerol phosphate/blood clot adhered to and was partly retained on the surfaces of the defect. At six months, defects that had been treated with chitosan-glycerol phosphate/blood were filled with significantly more hyaline repair tissue (p < 0.05) compared with control defects. Repair tissue from medial femoral condyle defects that had been treated with chitosan-glycerol phosphate/blood contained more cells and more collagen compared with control defects and showed complete restoration of glycosaminoglycan levels. CONCLUSIONS Solidification of a chitosan-glycerol phosphate/blood implant in microfracture defects improved cartilage repair compared with microfracture alone by increasing the amount of tissue and improving its biochemical composition and cellular organization.

In vitro osteogenesis assays: Influence of the primary cell source on alkaline phosphatase activity and mineralization

In trabecular bone fracture repair in vivo, osteogenesis occurs through endochondral ossification under hypoxic conditions, or through woven bone deposition in the vicinity of blood vessels. In vitro osteogenesis assays are routinely used to test osteoblastic responses to drugs, hormones, and biomaterials for bone and cartilage repair applications. These cell culture models recapitulate events that occur in woven bone synthesis, and are carried out using primary osteoblasts, osteoblast precursors such as bone marrow-derived mesenchymal stromal cells (BMSCs), or various osteoblast cell lines. With time in culture, cell differentiation is typically assessed by examining levels of alkaline phosphatase activity (an early osteoblast marker) and by evaluating the assembly of a collagen (type I)-containing fibrillar extracellular matrix that mineralizes. In this review, we have made a comparative analysis of published osteogenic assays using calvarial cells, calvaria-derived cell lines, and bone marrow stromal cells. In all of these cell types, alkaline phosphatase activity shows similar progression over time using a variety of osteogenic and mineralizing media conditions; however, levels of alkaline phosphatase activity are not proportional to observed mineralization levels.

Drilling and microfracture lead to different bone structure and necrosis during bone-marrow stimulation for cartilage repair

Bone marrow stimulation is performed using several surgical techniques that have not been systematically compared or optimized for a desired cartilage repair outcome. In this study, we investigated acute osteochondral characteristics following microfracture and comparing to drilling in a mature rabbit model of cartilage repair. Microfracture holes were made to a depth of 2 mm and drill holes to either 2 mm or 6 mm under cooled irrigation. Animals were sacrificed 1 day postoperatively and subchondral bone assessed by histology and micro‐CT. We confirmed one hypothesis that microfracture produces fractured and compacted bone around holes, essentially sealing them off from viable bone marrow and potentially impeding repair. In contrast, drilling cleanly removed bone from the holes to provide access channels to marrow stroma. Our second hypothesis that drilling would cause greater osteocyte death than microfracture due to heat necrosis was not substantiated, because more empty osteocyte lacunae were associated with microfracture than drilling, probably due to shearing and crushing of adjacent bone. Drilling deeper to 6 mm versus 2 mm penetrated the epiphyseal scar in this model and led to greater subchondral hematoma. Our study revealed distinct differences between microfracture and drilling for acute subchondral bone structure and osteocyte necrosis. Additional ongoing studies suggest these differences significantly affect long‐term cartilage repair outcome.

Meniscus structure in human, sheep, and rabbit for animal models of meniscus repair.

Meniscus injury is a frequently encountered clinical orthopedic issue and is epidemiologically correlated to osteoarthritis. The development of new treatments for meniscus injury is intimately related to the appropriateness of animal models for their investigation. The purpose of this study was to structurally compare human menisci to sheep and rabbit menisci to generate pertinent animal models for meniscus repair. Menisci were analyzed histologically, immunohistochemically, and by environmental scanning electron microscopy (ESEM). In all species, collagen I appeared throughout most menisci, but was absent from the inner portion of the tip in some samples. Collagen II was present throughout the inner main meniscal body, while collagen VI was found in pericellular and perivascular regions. The glycosaminoglycan‐rich inner portion of menisci was greater in area for rabbit and sheep compared to human. Cells were rounded in central regions and more fusiform at the surface, with rabbit being more cellular than sheep and human. Vascular penetration in rabbit was confined to the very outermost region (1% of meniscus length), while vessels penetrated deeper into sheep and human menisci (11–15%). ESEM revealed a lamellar collagenous structure at the articulating surfaces of sheep and human menisci that was absent in rabbit. Taken together, these data suggest that the main structural features that will influence meniscus repair—cellularity, vascularity, collagen structure—are similar in sheep and human but significantly different in rabbit, motivating the development of ovine meniscus repair models.

The Chondrocyte Cytoskeleton in Mature Articular Cartilage: Structure and Distribution of Actin, Tubulin, and Vimentin Filaments

We investigated the structure of the chondrocyte cytoskeleton in intact tissue sections of mature bovine articular cartilage using confocal fluorescence microscopy complemented by protein extraction and immunoblotting analysis. Actin microfilaments were present inside the cell membrane as a predominantly cortical structure. Vimentin and tubulin spanned the cytoplasm from cell to nuclear membrane, the vimentin network appearing finer compared to tubulin. These cytoskeletal structures were present in chondrocytes from all depth zones of the articular cartilage. However, staining intensity varied from zone to zone, usually showing more intense staining for the filament systems at the articular surface compared to the deeper zones. These results obtained on fluorescently labeled sections were also corroborated by protein contents extracted and observed by immunoblotting. The observed cytoskeletal structures are compatible with some of the proposed cellular functions of these systems and support possible microenvironmental regulation of the cytoskeleton, including that due to physical forces from load-bearing, which are known to vary through the depth layers of articular cartilage.

Depth of Subchondral Perforation Influences the Outcome of Bone Marrow Stimulation Cartilage Repair

Subchondral drilling and microfracture are bone marrow stimulation techniques commonly used for the treatment of cartilage defects. Few studies to date have examined the technical variants which may influence the success of the cartilage repair procedures. This study compared the effect of hole depth (6 mm vs. 2 mm) and hole type (drill vs. microfracture) on chondral defect repair using a mature rabbit model. Results from quantitative histomorphometry and histological scoring showed that deeper versus shallower drilling elicited a greater fill of the cartilage defect with a more hyaline character in the repair matrix indicated by significant improvement (p = 0.021) in the aggregate measure of increased cartilage defect fill, increased glycosaminoglycan and type II collagen content and reduced type I collagen content of total soft repair tissue. Compared to microfracture at the same 2 mm depth, drilling to 2 mm produced a similar quantity and quality of cartilage repair (p = 0.120) according to the aggregate indicator described above. We conclude that the depth of bone marrow stimulation can exert important influences on cartilage repair outcomes.

International Cartilage Repair Society (ICRS) Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials.

Cartilage repair strategies aim to resurface a lesion with osteochondral tissue resembling native cartilage, but a variety of repair tissues are usually observed. Histology is an important structural outcome that could serve as an interim measure of efficacy in randomized controlled clinical studies. The purpose of this article is to propose guidelines for standardized histoprocessing and unbiased evaluation of animal tissues and human biopsies. Methods were compiled from a literature review, and illustrative data were added. In animal models, treatments are usually administered to acute defects created in healthy tissues, and the entire joint can be analyzed at multiple postoperative time points. In human clinical therapy, treatments are applied to developed lesions, and biopsies are obtained, usually from a subset of patients, at a specific time point. In striving to standardize evaluation of structural endpoints in cartilage repair studies, 5 variables should be controlled: 1) location of biopsy/sample section, 2) timing of biopsy/sample recovery, 3) histoprocessing, 4) staining, and 5) blinded evaluation with a proper control group. Histological scores, quantitative histomorphometry of repair tissue thickness, percentage of tissue staining for collagens and glycosaminoglycan, polarized light microscopy for collagen fibril organization, and subchondral bone integration/structure are all relevant outcome measures that can be collected and used to assess the efficacy of novel therapeutics. Standardized histology methods could improve statistical analyses, help interpret and validate noninvasive imaging outcomes, and permit cross-comparison between studies. Currently, there are no suitable substitutes for histology in evaluating repair tissue quality and cartilaginous character.

Characterization of Subchondral Bone Repair for Marrow-Stimulated Chondral Defects and Its Relationship to Articular Cartilage Resurfacing

Background Microfracture and drilling are bone marrow–stimulation techniques that initiate cartilage repair by providing access to cell populations in subchondral bone marrow. This study examined the effect of hole depth and of microfracture versus drilling on subchondral bone repair and cartilage repair in full-thickness chondral defects. Hypotheses Repaired subchondral bone does not reconstitute its native structure and exhibits atypical morphologic features. Drilling deeper induces greater bone remodeling and is related to improved cartilage repair. Study Design Controlled laboratory study. Methods Trochlear cartilage defects debrided of the calcified layer were prepared bilaterally in 16 skeletally mature rabbits. Drill holes were made to a depth of 2 mm or 6 mm and microfracture holes to 2 mm. Animals were sacrificed 3 months postoperatively, and joints were scanned by micro–computed tomography before histoprocessing. Bone repair was assessed with a novel scoring system and by 3-dimentional micro–computed tomography and compared with intact controls. Correlation of subchon-dral bone features to cartilage repair outcome was performed. Results Although surgical holes were partly repaired with mineralized tissue, atypical features such as residual holes, cysts, and bony overgrowth were frequently observed. For all treatment groups, repair led to an average bone volume density similar to that of the controls but the repair bone was more porous and branched as shown by significantly higher bone surface area density and connectivity density. Deeper versus shallower drilling induced a larger region of repairing and remodeling subchondral bone that positively correlated with improved cartilage repair. Conclusion Incomplete reconstitution of normal bone structure and continued remodeling occurred in chondral defects 3 months after bone marrow stimulation. Deep drilling induced a larger volume of repairing and remodeling bone, which appeared beneficial for chondral repair. Clinical Relevance Bone marrow stimulation does not reconstitute normal bone structure. Strategies that increase subchondral bone involvement in marrow stimulation could further benefit cartilage repair.

Preclinical Studies for Cartilage Repair Recommendations from the International Cartilage Repair Society

Investigational devices for articular cartilage repair or replacement are considered to be significant risk devices by regulatory bodies. Therefore animal models are needed to provide proof of efficacy and safety prior to clinical testing. The financial commitment and regulatory steps needed to bring a new technology to clinical use can be major obstacles, so the implementation of highly predictive animal models is a pressing issue. Until recently, a reductionist approach using acute chondral defects in immature laboratory species, particularly the rabbit, was considered adequate; however, if successful and timely translation from animal models to regulatory approval and clinical use is the goal, a step-wise development using laboratory animals for screening and early development work followed by larger species such as the goat, sheep and horse for late development and pivotal studies is recommended. Such animals must have fully organized and mature cartilage. Both acute and chronic chondral defects can be used but the later are more like the lesions found in patients and may be more predictive. Quantitative and qualitative outcome measures such as macroscopic appearance, histology, biochemistry, functional imaging, and biomechanical testing of cartilage, provide reliable data to support investment decisions and subsequent applications to regulatory bodies for clinical trials. No one model or species can be considered ideal for pivotal studies, but the larger animal species are recommended for pivotal studies. Larger species such as the horse, goat and pig also allow arthroscopic delivery, and press-fit or sutured implant fixation in thick cartilage as well as second look arthroscopies and biopsy procedures.