A chondrocyte infiltrated collagen type I/III membrane (MACI® implant) improves cartilage healing in the equine patellofemoral joint model.

UNLABELLED: Autologous chondrocyte implantation (ACI) has improved outcome in long-term studies of joint repair in man. However, ACI requires sutured periosteal flaps to secure the cells, which precludes minimally-invasive implantation, and introduces complications with arthrofibrosis and graft hypertrophy. This study evaluated ACI on a collagen type I/III scaffold (matrix-induced autologous chondrocyte implantation; MACI(®)) in critical sized defects in the equine model. METHODS: Chondrocytes were isolated from horses, expanded and seeded onto a collagen I/III membrane (ACI-Maix™) and implanted into one of two 15-mm defects in the femoral trochlear ridge of six horses. Control defects remained empty as ungrafted debrided defects. The animals were examined daily, scored by second look arthroscopy at 12 weeks, and necropsy examination 6 months after implantation. Reaction to the implant was determined by lameness, and synovial fluid constituents and synovial membrane histology. Cartilage healing was assessed by arthroscopic scores, gross assessment, repair tissue histology and immunohistochemistry, cartilage glycosaminoglycan (GAG) and DNA assay, and mechanical testing. RESULTS: MACI(®) implanted defects had improved arthroscopic second-look, gross healing, and composite histologic scores, compared to spontaneously healing empty defects. Cartilage GAG and DNA content in the defects repaired by MACI implant were significantly improved compared to controls. Mechanical properties were improved but remained inferior to normal cartilage. There was minimal evidence of reaction to the implant in the synovial fluid, synovial membrane, subchondral bone, or cartilage. CONCLUSIONS: The MACI(®) implant appeared to improve cartilage healing in a critical sized defect in the equine model evaluated over 6 months.

Local gene delivery of heme oxygenase-1 by adeno-associated virus into osteoarthritic mouse joints exhibiting synovial oxidative stress.

OBJECTIVE: To evaluate the role of synovial oxidative stress on joint pathology in a spontaneous mouse model of osteoarthritis (OA) by intra-articular (IA) delivery of recombinant adeno-associated virus (rAAV) expressing anti-oxidant protein heme oxygenase-1 (HO-1). METHODS: Joint transduction by rAAV vectors was evaluated with serotype 1, 2, 5 and 8 capsids carrying LacZ gene administered by IA injections into STR/ort mice. Transduced cell types were identified by ß-galactosidase staining in sectioned joints. Effect of oxidative stress on AAV transduction of primary synoviocytes in vitro was quantitated by fluorescence-activated cell sorting (FACS) analysis. In vivo, the efficacy of rAAV1/HO-1 was tested by IA administration into STR/ort mice followed by histopathological scoring of cartilage. Levels of 3-nitrotyrosine (3-NT) and HO-1 were assessed by immunohistochemistry (IHC) of joint sections. RESULTS: Administration of a rAAV1 based vector into OA mouse joints resulted in transduction of the synovium, joint capsule, adipocytes and skeletal muscle while none of the serotypes showed significant cartilage transduction. All OA joints exhibited significantly elevated levels of oxidative stress marker, 3-NT, in the synovium compared to OA-resistant CBA-strain of mice. In vitro studies demonstrated that AAV transgene expression in primary synoviocytes was augmented by oxidative stress induced by H(2)O(2) and that a rAAV expressing HO-1 reduced the levels of oxidative stress. In vivo, HO-1 was increased in the synovium of STR/ort mice. However, delivery of rAAV1/HO-1 into OA joints did not reduce cartilage degradation. CONCLUSIONS: AAV-mediated HO-1 delivery into OA joints during active disease was not sufficient to improve cartilage pathology in this model.

Hylan G-F 20 maintains cartilage integrity and decreases osteophyte formation in osteoarthritis through both anabolic and anti-catabolic mechanisms.

OBJECTIVE: To perform a molecular mechanism-based investigation of the chondroprotective potential of hylan G-F 20. METHOD: The effects of hylan G-F 20 on IL-1ß-induced glycosaminoglycan (GAG) depletion and matrix metalloproteinase (MMP) expression in bovine and human cartilage explants were evaluated. Three weekly intra-articular hylan G-F 20 or control injections were administered 4 weeks post-operatively to rabbits with surgically induced osteoarthritis (OA). Cartilage histopathologic scores and osteophyte size were evaluated at 1, 4, and 8 weeks post-injections. Histomorphometry and immunostaining were used to quantify cartilage area and type II collagen (Col II) intensity, and real-time polymerase chain reaction (PCR) was used to examine the mRNA levels of Col2A1, MMP-13, -16 and IL-1ß at 1 week. RESULTS: Hylan G-F 20 retained GAG in IL-1ß-exposed bovine and human cartilage explants and abrogated IL-1ß-mediated increases in MMP-1, -3, and -13 in human explant culture. Hylan G-F 20‒treated OA joints had significantly better cartilage integrity at 1 and 4 weeks post-treatment and significantly smaller osteophytes at 4 weeks compared with control. Col2A1 mRNA increased with hylan G-F 20 treatment, which correlated with a trend toward increased Col II immunostaining. MMP-13 and -16 mRNAs increased in OA cartilage, but were not significantly altered by hylan G-F 20. IL-1ß mRNA was undetectable in cartilage and unaltered in the synovium. CONCLUSIONS: Hylan G-F 20 improved cartilage integrity and decreased osteophyte formation in the rabbit model of OA. Our results suggest that hylan G-F 20 may stimulate cartilage repair by increasing Col II, and inhibit IL-1ß-mediated matrix degradation by decreasing MMPs.

TGF beta 1 and biglycan, decorin, and fibromodulin metabolism in canine cartilage.

OBJECTIVE: Small proteoglycans (PGs) may accumulate in late stage osteoarthritis even as aggrecan is lost. It is not clear what role transforming growth factor (TGF) beta has in this accumulation. Our goal was to investigate the ability of TGF beta 1 to modulate the synthesis and accumulation of decorin, biglycan, and fibromodulin in cartilage explants cultured under conditions in which aggrecan synthesis remains relatively constant. DESIGN: Articular cartilage was cultured in the presence or absence of 4 ng/ml TGF beta 1 for up to 16 days. Material extracted from cartilage was assayed for 35SO(4)-large and small PGs and for total endogenous decorin, biglycan and fibromodulin. RESULTS: The synthesis of 35SO(4)-small PGs increased during the 16 days in culture in response to TGF beta 1, but declined in control cultures. The difference in 35SO(4)-decorin between TGF beta 1 and control samples reached nine-fold after 16 days, while the difference in total endogenous decorin was less than 1.5-fold. 35SO(4)-decorin, which was present in TGF beta 1-treated cultures had an identical core protein, but a longer glycosaminoglycan chain than that of decorin in control cultures. No significant differences in endogenous biglycan were detected, but accumulation of fibromodulin in TGF beta 1 explants exceeded fibromodulin in controls, on average, by 3.8-fold. Fibromodulin was present in cartilage in both keratan sulfate- and non-sulfated oligosaccharide-substituted forms. CONCLUSIONS: The accumulation of each of the three small PGs was affected to a different extent in response to TGF beta 1. Of the three, fibromodulin content was most rapidly augmented in response to TGF beta 1.

Effect of dimethylsulfoxide on articular cartilage proteoglycan synthesis and degradation, chondrocyte viability, and matrix water content.

OBJECTIVE: To determine the effects of dimethylsulfoxide (DMSO) exposure on cartilage proteoglycan (PG) synthesis, PG degradation, chondrocyte viability, and matrix water content. STUDY DESIGN: Using a cartilage explant culture system, PG synthesis, PG degradation, matrix water content, and chondrocyte viability were determined for cartilage exposed to DMSO daily for selected periods of time. ANIMALS OR SAMPLE POPULATION: Juvenile bovine (calf) carpometacarpal joint cartilage explants. METHODS: PG synthesis: Explants (n = 30/group) were separated into 10 groups based on the time of daily exposure to 10% DMSO. Exposure time was repeated daily for 3 days. The control group was incubated in basal medium alone for 3 days, with daily medium changes. Once all DMSO exposure times were complete for the third day, PG synthesis was determined by analysis of incorporation of radiolabelled sulfate. Cell viability: Explants (n = 3/group) were subjected to an identical DMSO exposure protocol, and examined histologically. The percentage of viable cells/high power field (hpf) was calculated for each group. PG degradation: Explants (n = 21/group) were preincubated with radiolabelled sulfate, then subjected to a similar DMSO exposure protocol. The medium was collected from all explants daily and assayed for PG content. After 3 days, the explants were digested and total labelled PG content determined. Percent of total explant labelled PG content released into the medium daily was determined for each group. Water content: Explants (n = 21/group) were separated into three treatment groups, one of which had no treatments performed, whereas the other two groups were incubated in basal medium for 72 hours, one with, and one without, 10% DMSO. Wet and dry weights were determined, and percent water calculated, for all three groups. Separate 1-way ANOVA were performed, with appropriate post hoc tests (P < .05). RESULTS: PG synthesis was significantly lower than control for all time periods of DMSO exposure except for 1 and 3 hours, and decreased in a time-dependent manner after the 1-hour exposure time. The mean percentage of viable cells/hpf was significantly lower than control for the 1-, 3-, 9-, 12-, and 24-hour treatment groups. There was no significant difference in PG degradation for any group compared with control for the first 2 days of incubation. All groups except the 24-hour group had a significantly higher degradation compared with control for the third day of incubation. Cartilage exposed to DMSO for 72 hours had a significantly lower water content, and cartilage incubated in basal medium alone for 72 hours had a significantly higher water content than cartilage that received no DMSO and no incubation. CONCLUSIONS: DMSO, in relatively low concentration, is detrimental to articular cartilage PG synthesis in a time-dependent manner. Dehydration of the cartilage and chondrocyte death also occur with increasing time of DMSO exposure. Significant PG degradation occurs on the third day of culture with daily DMSO exposure. CLINICAL RELEVANCE: As a joint lavage solution, DMSO has potentially deleterious effects on the metabolism of chondrocytes.

Oocyte transfer in mares.

Oocytes were collected from dominant preovulatory follicles of donor mares 24 hours after administration of human chorionic gonadotropin. Oocytes were incubated in vitro for 12 or 18 hours before transfer to recipient mares, representing maturation times after human chorionic gonadotropin administration of 36 and 42 hours, respectively. Pregnancy rates after transfer were 4 of 5 in the 36-hour group and 2 of 3 in the 42-hour group. The overall pregnancy rate achieved (6/8 mares) indicated that oocyte transfer may be useful clinically.

Effects of tendon grip technique (frozen versus unfrozen) on in vitro surface strain measurements of the equine deep digital flexor tendon.

OBJECTIVE: To determine effects of tendon grip technique on in vitro surface strain measurements of equine deep digital flexor tendon (DDFT) when loaded in tension. SAMPLE POPULATION: 12 hind limb DDFT from 8 adult horses (mean age, 9.8 years [range, 4.5 to 17 years]; mean body weight, 472 kg [range, 450 to 509 kg]), with no clinical evidence of hind limb lameness. DESIGN AND PROCEDURE: After calibration, liquid mercury strain gauges were sutured to plantar surfaces of the tendons at distal (position 1), middle (position 2), and proximal (position 3) metatarsal regions. Each tendon was affixed to a materials testing machine (distally by the distal phalanx, and proximally by a metal clamp), and loaded once in tension, at a strain rate of 2.8 to 3.0%/s, to 3% clamp displacement. Liquid nitrogen was used to freeze the proximal ends of 6 tendons before placement in the clamp. Proximal ends of the remaining 6 tendons were left unfrozen. Surface strains were determined with the aid of low-resistance bridge circuits. Data were analyzed at time points corresponding to 1, 2, and 3% clamp displacement. RESULTS: Mean surface strains of tendons with frozen ends were significantly greater than those for tendons with unfrozen ends, and closer to strain measured by clamp displacement, at 1 and 2% clamp displacement. This difference was present but not significant at 3% clamp displacement. Mean local surface strain was not significantly different between the 3 gauge positions. CONCLUSIONS: Freezing tendon ends is a useful technique to increase measured surface strains in equine DDFT, thereby making them closer to universal strain as measured by clamp displacement, and, thus, more likely to represent true surface strain.