Mark B. Hurtig

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.

OP-1/BMP-7 in cartilage repair

Three years ago we published a book chapter on the role of bone morphogenetic proteins (BMPs) in cartilage repair. Since that time our understanding of the function of osteogenic protein-1 (OP-1) or BMP-7 in cartilage homeostasis and repair has substantially improved and therefore we decided to devote a current review solely to this BMP. Here we summarise the information accumulated on OP-1 from in vitro and ex vivo studies with cartilage cells and tissues as well as from in vivo studies of cartilage repair in various animal models. The primary focus is on articular chondrocytes and cartilage, but data will also be presented on nonarticular cartilage, particularly from the intervertebral disc. The data show that OP-1 is a unique growth factor which, unlike other members of the same BMP family, exhibits in addition to its strong pro-anabolic activity very prominent anti-catabolic properties. Animal studies have demonstrated that OP-1 has the ability to repair cartilage in vivo in various models of articular cartilage degradation, including focal osteochondral and chondral defects and osteoarthritis, as well as models of degeneration in intervertebral disc cartilage. Together our findings indicate a significant promise for OP-1 as therapeutic in cartilage repair.RésuméIl y a trois ans a été publié le chapitre d’un livre sur le rôle des BMP dans la réparation cartilagineuse. Depuis cette époque, notre compréhension du mécanisme d’action de l’OP-1 ou BMP-7 dans la régulation cartilagineuse et sa réparation ont été améliorées. Nous avons décidé de passer en revue les différents travaux portant sur cette BMP. Nous avons résumé les informations accumulées sur l’OP-1, les études in-vitro et ex-vivo sur les cellules cartilagineuses et sur les tissus à partir de travaux sur la réparation cartilagineuse chez différents modèles d’animaux. Ces travaux ont été essentiellement centrés sur les chondrocytes du cartilage articulaire mais l’on peut également trouver des données sur le cartilage non articulaire particulièrement au niveau du disque inter vertébral. Ces données montrent que l’OP-1 est le seul facteur de croissance. Dans la famille des autres BMP les activités pro-anaboliques sont plus importantes que les propriétés anti anaboliques. Les études animales démontrent que l’OP-1 permet une réparation cartilagineuse parmi les différents modèles de lésions de ce cartilage, outre les lésions cartilagineuses, les lésions d’arthrose et les lésions de dégénérescence des disques intervertébraux. Tout cela nous fait entrevoir une sérieuse avancée thérapeutique grâce à l’OP-1 dans les champs des réparations cartilagineuses.

Role of cartilage collagen fibrils networks in knee joint biomechanics under compression.

Collagen fibrils networks in knee cartilage and menisci change in content and structure from a region to another. While resisting tension, they influence global joint response as well as local strains particularly at short-term periods. To investigate the role of fibrils networks in knee joint mechanics and in particular cartilage response, a novel model of the knee joint is developed that incorporates the cartilage and meniscus fibrils networks as well as depth-dependent properties in cartilage. The joint response under up to 2000N compression is investigated for conditions simulating the absence in cartilage of deep fibrils normal to subchondral bone or superficial fibrils parallel to surface as well as localized split of cartilage at subchondral junction or localized damage to superficial fibrils at loaded areas. Deep vertical fibrils network in cartilage play a crucial role in stiffening (by 10%) global response and protecting cartilage by reducing large strains (from maximum of 102% to 38%), in particular at subchondral junction. Superficial horizontal fibrils protect the tissue mainly from excessive strains at superficial layers (from 27% to 8%). Local cartilage split at base disrupts the normal function of vertical fibrils at the affected areas resulting in higher strains. Deep fibrils, and to a lesser extent superficial fibrils, play dominant mechanical roles in cartilage response under transient compression. Any treatment modality attempting to repair or regenerate cartilage defects involving partial or full thickness osteochondral grafts should account for the crucial role of collagen fibrils networks and the demanding mechanical environment of the tissue.

The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the horse.

OBJECTIVE Equine models of osteoarthritis (OA) have been used to investigate pathogenic pathways of OA and evaluate therapeutic candidates for naturally occurring equine OA which is a significant clinical disease in the horse. This review focuses on the macroscopic and microscopic criteria for assessing naturally occurring OA in the equine metacarpophalangeal joint as well as the osteochondral fragment-exercise model of OA in the equine middle carpal joint. METHODS A review was conducted of all published OA studies using horses and the most common macroscopic and microscopic scoring systems were summarized. Recommendations regarding methods of OA assessment in the horse have been made based on published studies. RESULTS A modified Mankin scoring system is recommended for semi-quantitative histological assessment of OA in horses due to its already widespread use and similarity to other scoring systems. Recommendations are also provided for histological scoring of synovitis and macroscopic lesions of OA as well as changes in the calcified cartilage and subchondral bone of naturally occurring OA. CONCLUSIONS The proposed system for assessment of equine articular tissues provides a useful method to quantify OA change. It is believed that addition of quantitative tracing onto plastic and macroscopic measurement as recently described would be an improvement for overall assessment of articular cartilage change.

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.

Protection against LPS-induced cartilage inflammation and degradation provided by a biological extract of Mentha spicata

BackgroundA variety of mint [Mentha spicata] has been bred which over-expresses Rosmarinic acid (RA) by approximately 20-fold. RA has demonstrated significant anti-inflammatory activity in vitro and in small rodents; thus it was hypothesized that this plant would demonstrate significant anti-inflammatory activity in vitro. The objectives of this study were: a) to develop an in vitro extraction procedure which mimics digestion and hepatic metabolism, b) to compare anti-inflammatory properties of High-Rosmarinic-Acid Mentha spicata (HRAM) with wild-type control M. spicata (CM), and c) to quantify the relative contributions of RA and three of its hepatic metabolites [ferulic acid (FA), caffeic acid (CA), coumaric acid (CO)] to anti-inflammatory activity of HRAM.MethodsHRAM and CM were incubated in simulated gastric and intestinal fluid, liver microsomes (from male rat) and NADPH. Concentrations of RA, CA, CO, and FA in simulated digest of HRAM (HRAMsim) and CM (CMsim) were determined (HPLC) and compared with concentrations in aqueous extracts of HRAM and CM. Cartilage explants (porcine) were cultured with LPS (0 or 3 μg/mL) and test article [HRAMsim (0, 8, 40, 80, 240, or 400 μg/mL), or CMsim (0, 1, 5 or 10 mg/mL), or RA (0.640 μg/mL), or CA (0.384 μg/mL), or CO (0.057 μg/mL) or FA (0.038 μg/mL)] for 96 h. Media samples were analyzed for prostaglandin E2 (PGE2), interleukin 1β (IL-1), glycosaminoglycan (GAG), nitric oxide (NO) and cell viability (differential live-dead cell staining).ResultsRA concentration of HRAMsim and CMsim was 49.3 and 0.4 μg/mL, respectively. CA, FA and CO were identified in HRAMsim but not in aqueous extract of HRAM. HRAMsim (≥ 8 μg/mL) inhibited LPS-induced PGE2 and NO; HRAMsim (≥ 80 μg/mL) inhibited LPS-induced GAG release. RA inhibited LPS-induced GAG release. No anti-inflammatory or chondroprotective effects of RA metabolites on cartilage explants were identified.ConclusionsOur biological extraction procedure produces a substance which is similar in composition to post-hepatic products. HRAMsim is an effective inhibitor of LPS-induced inflammation in cartilage explants, and effects are primarily independent of RA. Further research is needed to identify bioactive phytochemical(s) in HRAMsim.

Mapping of donor and recipient site properties for osteochondral graft reconstruction of subchondral cystic lesions in the equine stifle joint.

REASONS FOR PERFORMING STUDY To improve osteochondral graft reconstruction of subchondral cystic lesions in the medial and lateral femoral condyles by matching the material properties of donor and recipient sites. OBJECTIVES To measure biomechanical and biochemical parameters that influence the function and healing of osteochondral grafts used to reconstruct subchondral cystic lesions. HYPOTHESIS Suitable donor sites are available within the stifle joint for reconstructing the femoral condyles, despite considerable regional property variation. METHODS Fifty-six osteochondral cores were harvested from 6 distal femurs for initial studies that determined subchondral bone modulus of elasticity and ultimate stress. In a second study, 28 osteochondral cores were harvested from 6 distal femurs to measure cartilage aggregate modulus, thickness and sulphated glycosaminoglycan (sGAG) content. Using micro-CT imaging, subchondral bone mineral density and bone volume fraction were also measured. In both studies 2-dimensional contour plots using a bicubic interpolation method and normalised data were generated to allow visual comparison of joint surface characteristics. Statistical comparisons between donor and recipient site raw data were made using an ANOVA for repeated measures with a post hoc Tukey test. RESULTS Material properties of cartilage and bone vary considerably over the surface of the stifle joint but the central region of the medial condyle, where subchondral cystic lesions freqdently occur, typically demonstrated bone strength and modulus values of the highest observed. Cartilage thickness and aggregate modulus were highest in the medial femoral condyle and axial aspect of the lateral condyle. CONCLUSIONS Material properties of the grafts from the trochlear groove and axial aspect of the lateral trochlear ridge were the closest match for those found in the medial condyle, whereas properties of the lateral condyle were most similar to those found in the trochlear groove and axial aspect of the medial trochlear ridge.

Serum non-coding RNAs as biomarkers for osteoarthritis progression after ACL injury

OBJECTIVE The aim of this study was to examine serum non-coding RNAs as potential biomarkers for cartilage damage associated with anterior cruciate ligament (ACL) injury. METHODS Serum was obtained from 80 patients 1 year after surgery for ACL injury and 60 normal donors without overt skeletal injury. Total serum RNA was isolated, small non-coding RNAs profiled by TaqMan array MicroRNA (miRNA) analysis and individual small RNA assays performed by quantitative TaqMan RT-PCR (qPCR). Semi-quantitative magnetic resonance imaging (MRI) analysis was performed using Whole Organ Magnetic Resonance Knee Score (WORMS) scoring for analysis of cartilage damage. RESULTS Initial TaqMan array miRNA profiling showed an increased serum concentration of a small nucleolar RNA (snoRNA), U48, in five patients with cartilage damage compared with that in five patients without cartilage damage and six normal donors. Independent qPCR analysis of snoRNAs in serum from all patients and normal donors showed a strong association between the serum level of another snoRNA, U38, and cartilage damage in ACL injury patients and together with snoRNA, U48, clear distinction between ACL injury patients and normal donors. CONCLUSION SnoRNAs U38 and U48 are significantly elevated in the serum of patients developing cartilage damage at 1 year after ACL injury. Serum levels of U38 have the potential to facilitate early diagnosis of patients with cartilage damage after ACL injury. This study suggests serum non-coding RNAs may serve as novel noninvasive biomarkers for the detection and assessment of cartilage damage after ACL injury.

Multiple Pathways to Osteoarthritis and Articular Fractures: Is Subchondral Bone the Culprit?

Osteoarthritis and articular fractures are commonly responsible for early retirement from athletic performance. The subchondral bone (SCB) in those conditions is being recognized as an integral component in their pathophysiology. Early recognition of these potentially career-ending diseases may require understanding of the progression of changes occurring in SCB with time and exercise.

Mechanical properties of subchondral bone in the distal aspect of third metacarpal bones from Thoroughbred racehorses

OBJECTIVE To characterize the mechanical properties of subchondral bone (SCB) of the distopalmar aspect of the condyles of the third metacarpal bone (MC3) and their correlations with structural aspects of MC3s in Thoroughbred racehorses. SAMPLE POPULATION 12 pairs of MC3s from Thoroughbred racehorses euthanized for various reasons. PROCEDURES MC3s were collected from horses with mild (n = 6) and with severe (6) SCB changes, as determined by micro-computed tomography (CT). Specimens of SCB plate and trabecular bone were cut from the distopalmar aspect of condyles and sagittal ridge and examined with 3-dimensional micro-CT. Specimens were tested in compression, and elastic modulus, yield stress, yield strain, and toughness were calculated. Apparent and true bone mineral density, bone volume fraction, trabecular thickness, trabecular separation, and connectivity were also calculated. Differences in mechanical properties among various classifications of bone were evaluated. Correlations between structural and mechanical variables were also assessed. RESULTS No differences were detected between left and right forelimbs. Specimens from condyles had higher values for elastic modulus, yield stress, and toughness than did specimens of sagittal ridge. In SCB with severe changes attributable to SCB disease, SCB plate was weaker and trabecular bone was stronger than in SCB with mild changes. Microstructural and mechanical properties were significantly correlated. CONCLUSIONS AND CLINICAL RELEVANCE A marked gradient in mechanical properties of SCB from horses, which could be involved in the pathogenesis of condylar fractures, was detected. Mechanical properties of SCB from the distal aspect of MC3s can be predicted to some extent via micro-CT.