A pilot study to determine the optimal dose of scAAVIL-1ra in a large animal model of post-traumatic osteoarthritis.

Gene therapy approaches using adeno-associated viral vectors have been successfully tested in the equine post-traumatic osteoarthritis (PTOA) model. Owing to differences in the levels of transgene expression and adverse tissue reactions observed in published studies, we sought to identify a safe therapeutic dose of scAAVIL-1ra in an inflamed and injured joint that would result in improved functional outcomes without any adverse events. scAAVIL-1ra was delivered intra-articularly over a 100-fold range, and horses were evaluated throughout and at the end of the 10-week study. A dose-related increase in IL-1ra levels with a decrease in PGE2 levels was observed, with the peak IL-1ra concentration being observed 7 days post-treatment in all groups. Perivascular infiltration with mononuclear cells was observed within the synovial membrane of the joint treated with the highest viral dose of 5 × 1012 vg, but this was absent in the lower-dosed joints. The second-highest dose of scAAVeqIL-1ra 5 × 1011 vg demonstrated elevated IL-1ra levels without any cellular response in the synovium. Taken together, the data suggest that the 10-fold lower dose of 5 × 1011vg scAAVIL-1ra would be a safe therapeutic dose in an equine model of PTOA.

Longitudinal changes in tibial and femoral cartilage thickness are associated with baseline ambulatory kinetics and cartilage oligomeric matrix protein (COMP) measures in an asymptomatic aging population.

OBJECTIVE: To address the need for early knee osteoarthritis (OA) markers by testing if longitudinal cartilage thickness changes are associated with specific biomechanical and biological measures acquired at a baseline test in asymptomatic aging subjects. DESIGN: Thirty-eight asymptomatic subjects over age 45 years were studied at baseline and at an average of 7-9 year follow-up. Gait mechanics and knee MRI were measured at baseline and MRI was obtained at follow-up to assess cartilage thickness changes. A subset of the subjects (n = 12) also had serum cartilage oligomeric matrix protein measured at baseline in response to a mechanical stimulus (30-min walk) (mCOMP). Baseline measures, including the knee extension (KEM), flexion (KFM), adduction (KAM) moments and mCOMP, were tested for associations with cartilage thickness changes in specific regions of the knee. RESULTS: Cartilage change in the full medial femoral condyle (p = 0.005) and external medial femoral region (p = 0.041) was negatively associated with larger early stance peak KEM. Similarly, cartilage change in the full medial femoral region (p = 0.009) and medial femoral external region (p = 0.043) was negatively associated with larger first peak KAM, while cartilage change in the anterior medial tibia was positively associated with larger first peak KAM (p = 0.003). Cartilage change in the anterior medial tibia was also significantly associated (p = 0.011) with mCOMP levels 5.5-h post-activity (percentage of pre-activity levels). CONCLUSIONS: Interactions found between gait, mechanically-stimulated serum biomarkers, and cartilage thickness in an at-risk aging asymptomatic population suggest the opportunity for early detection of OA with new approaches that bridge across disciplines and scales.

Classification systems for platelet-rich plasma.

There is good scientific rationale to support the use of growth factors to promote musculoskeletal tissue regeneration. However, the clinical effectiveness of platelet-rich plasma (PRP) and other blood-derived products has yet to be proven. Characterization and reporting of PRP preparation protocols utilized in clinical trials for the treatment of musculoskeletal disease is highly inconsistent, and the majority of studies do not provide sufficient information to allow the protocols to be reproduced. Furthermore, the reporting of blood-derived products in orthopaedics is limited by the multiple PRP classification systems available, which makes comparison of results between studies challenging. Several attempts have been made to characterize and classify PRP; however, no consensus has been reached, and there is lack of a comprehensive and validated classification. In this annotation, we outline existing systems used to classify preparations of PRP, highlighting their advantages and limitations. There remains a need for standardized universal nomenclature to describe biological therapies, as well as a comprehensive and reproducible classification system for autologous blood-derived products. Cite this article: Bone Joint J 2019;101-B:891-896.

MRI UTE-T2* profile characteristics correlate to walking mechanics and patient reported outcomes 2 years after ACL reconstruction.

OBJECTIVE: Quantitative magnetic resonance imaging (MRI) ultrashort echo time (UTE) T2* is sensitive to cartilage deep tissue matrix changes after anterior cruciate ligament reconstruction (ACLR). This study was performed to determine whether UTE-T2* profile analysis is a useful clinical metric for assessing cartilage matrix degeneration. This work tests the hypotheses that UTE-T2* depthwise rates of change (profile slopes) correlate with clinical outcome metrics of walking mechanics and patient reported outcomes (PRO) in patients 2 years after ACLR. DESIGN: Thirty-six patients 2 years after ACLR completed knee MRI, gait analysis, and PRO. UTE-T2* maps were generated from MRI images and depthwise UTE-T2* profiles were calculated for weight-bearing cartilage in the medial compartment. UTE-T2* profiles from 14 uninjured subjects provided reference values. UTE-T2* profile characteristics, including several different measures of profile slope, were tested for correlation to kinetic and kinematic measures of gait and also to PRO. RESULTS: Decreasing UTE-T2* profile slopes in ACLR knees moderately correlated with increasing knee adduction moments (r = 0.41, P < 0.015), greater external tibial rotation (r = 0.44, P = 0.007), and moderately negatively correlated with PRO (r = -0.36, P = 0.032). UTE-T2* profiles from both ACLR and contralateral knees of ACLR subjects differed from that of uninjured controls (P < 0.015). CONCLUSIONS: The results of this study suggest that decreasing UTE-T2* profile slopes reflect cartilage deep tissue collagen matrix disruption in a population at increased risk for knee osteoarthritis (OA). That UTE-T2* profiles were associated with mechanical and patient reported measures of clinical outcomes support further study into a potential mechanistic relationship between these factors and OA development.

T2 texture index of cartilage can predict early symptomatic OA progression: data from the osteoarthritis initiative.

OBJECTIVE: There is an interest in using Magnetic Resonance Imaging (MRI) to identify pre-radiographic changes in osteoarthritis (OA) and features that indicate risk for disease progression. The purpose of this study is to identify image features derived from MRI T2 maps that can accurately predict onset of OA symptoms in subjects at risk for incident knee OA. METHODS: Patients were selected from the Osteoarthritis Initiative (OAI) control cohort and incidence cohort and stratified based on the change in total Western Ontario and McMaster Universities Arthritis (WOMAC) score from baseline to 3-year follow-up (80 non-OA progression and 88 symptomatic OA progression patients). For each patient, a series of image texture features were measured from the baseline cartilage T2 map. A linear discriminant function and feature reduction method was then trained to quantify a texture metric, the T2 texture index of cartilage (TIC), based on 22 image features, to identify a composite marker of T2 heterogeneity. RESULTS: Statistically significant differences were seen in the baseline T2 TIC between the non-progression and symptomatic OA progression populations. The baseline T2 TIC differentiates subjects that develop worsening of their WOMAC score OA with an accuracy between 71% and 76%. The T2 TIC differences were predominantly localized to a dominant knee compartment that correlated with the mechanical axis of the knee. CONCLUSION: Baseline heterogeneity in cartilage T2 as measured with the T2 TIC index is able to differentiate and predict individuals that will develop worsening of their WOMAC score at 3-year follow-up.

UTE-T2∗ mapping detects sub-clinical meniscus injury after anterior cruciate ligament tear.

OBJECTIVE: Meniscus tear is a known risk factor for osteoarthritis (OA). Quantitative assessment of meniscus degeneration, prior to surface break-down, is important to identification of early disease potentially amenable to therapeutic interventions. This work examines the diagnostic potential of ultrashort echo time-enhanced T2∗ (UTE-T2∗) mapping to detect human meniscus degeneration in vitro and in vivo in subjects at risk of developing OA. DESIGN: UTE-T2∗ maps of 16 human cadaver menisci were compared to histological evaluations of meniscal structural integrity and clinical magnetic resonance imaging (MRI) assessment by a musculoskeletal radiologist. In vivo UTE-T2∗ maps were compared in 10 asymptomatic subjects and 25 ACL-injured patients with and without concomitant meniscal tear. RESULTS: In vitro, UTE-T2∗ values tended to be lower in histologically and clinically normal meniscus tissue and higher in torn or degenerate tissue. UTE-T2∗ map heterogeneity reflected collagen disorganization. In vivo, asymptomatic meniscus UTE-T2∗ values were repeatable within 9% (root-mean-square average coefficient of variation). Posteromedial meniscus UTE-T2∗ values in ACL-injured subjects with clinically diagnosed medial meniscus tear (n=10) were 87% higher than asymptomatics (n=10, P<0.001). Posteromedial menisci UTE-T2∗ values of ACL-injured subjects without concomitant medial meniscal tear (n=15) were 33% higher than asymptomatics (P=0.001). Posterolateral menisci UTE-T2∗ values also varied significantly with degree of joint pathology (P=0.001). CONCLUSION: Significant elevations of UTE-T2∗ values in the menisci of ACL-injured subjects without clinical evidence of subsurface meniscal abnormality suggest that UTE-T2∗ mapping is sensitive to sub-clinical meniscus degeneration. Further study is needed to determine whether elevated subsurface meniscus UTE-T2∗ values predict progression of meniscal degeneration and development of OA.

Single intra-articular injection of adeno-associated virus results in stable and controllable in vivo transgene expression in normal rat knees.

OBJECTIVE: To test the hypothesis that in vivo transgene expression mediated by single intra-articular injection of adeno-associated virus serotype 2 (AAV2) persists within intra-articular tissues 1 year post-injection and can be externally controlled using an AAV2-based tetracycline-inducible gene regulation system containing the tetracycline response element (TRE) promoter. METHODS: Sprague Dawley rats received intra-articular injections of AAV2-cytomegalovirus (CMV)-enhanced green fluorescent protein (GFP) and AAV2-CMV-luciferase (Luc) into their right and left knees, respectively. Luciferase expression was evaluated over 1 year using bioluminescence imaging. After sacrifice, tissues were analyzed for GFP+ cells by fluorescent microscopy. To study external control of intra-articular AAV-transgene expression, another set of rats was co-injected with AAV2-TRE-Luc and AAV2-CMV-reverse-tetracycline-controlled transactivator (rtTA) into the right knees, and AAV2-CMV-Luc and AAV2-CMV-rtTA into the left knees. Rats received oral doxycycline (Dox), an analog of tetracycline, for 7 days. Luciferase expression was assessed by bioluminescence imaging. RESULTS: Luciferase expression was localized to the injected joint and persisted throughout the 1-year study period. Abundant GFP+ cells were observed within intra-articular soft tissues. Transgene expression in AAV2-TRE-Luc injected joints was upregulated by oral administration of Dox, and downregulated following its removal, at 14 days and 13 months post-AAV injection. CONCLUSIONS: This longitudinal in vivo study shows that sustained and stable AAV-mediated intra-articular transgene expression can be achieved through a single intra-articular injection and can be controlled using a tetracycline-controlled inducible AAV system in a normal rat knee model. Highly regulatable long-term intra-articular transgene expression is of potential clinical utility for development of treatment strategies for chronic intra-articular disease processes such as inflammatory and degenerative arthritis.

UTE-T2∗ mapping of human articular cartilage in vivo: a repeatability assessment.

INTRODUCTION: Ultrashort echo-time enhanced T2∗ (UTE-T2∗) mapping of articular cartilage is a novel quantitative MRI technique with the potential to visualize deep cartilage characteristics better than standard T2 mapping. The feasibility and intersession repeatability of UTE-T2∗ mapping of cartilage in vivo has not previously been evaluated. METHODS: Eleven asymptomatic subjects underwent repeat UTE-T2∗ imaging on a whole-body 3T MRI scanner on three consecutive days. Full-thickness, superficial and deep regions of interest (ROIs) were evaluated in the central weight-bearing zones of the medial femoral condyle (cMFC) and tibial plateau (cMTP). Intersession precision error across subjects was evaluated by the root-mean-square average coefficients of variation (RMSA-CV) and by the median of intra-subject standard deviations (SDs) of UTE-T2∗ values in each ROI. RESULTS: UTE-T2∗ values in vivo were found to be repeatable with relative (RMSA-CV) intersession precision errors of 8%, 6%, 16% for full-thickness, superficial and deep cMFC ROIs, corresponding to absolute errors (SD) of 1.2, 1.5, 1.5 ms, respectively. In cMTP tissue, UTE-T2∗ relative repeatability was 8%, 8%, 13%, corresponding to absolute repeatability of 1.0, 1.5, 2.1 ms (full-thickness, superficial, deep). UTE-T2∗ values were higher in superficial cartilage compared to deep in both cMFC (P≪0.001) and cMTP (P=0.0004) regions. CONCLUSION: In vivo 3D UTE-T2∗ mapping at 3T is feasible and can be implemented using a standard clinical MRI scanner and knee coil. Intersession precision error of UTE-T2∗ values in full-thickness ROIs in the weight-bearing regions of asymptomatic subjects is under 1.2 ms or 8% (RMSA-CV). Significant zonal and regional variations of UTE-T2∗ were seen.

The association of osteoarthritis risk factors with localized, regional and diffuse knee pain.

OBJECTIVE: To identify determinants of different patterns of knee pain with a focus on risk factors for knee osteoarthritis (OA). DESIGN: The Knee Pain Map is an interviewer-administered assessment that asks subjects to characterize their knee pain as localized, regional, or diffuse. A total of 2677 participants from the Osteoarthritis Initiative were studied. We used multinomial logistic regression to examine the relationship between risk factors for OA and knee pain patterns. We examined the bivariate and multivariate relationships of knee pain pattern with age, body mass index (BMI), sex, race, family history of total joint replacement, knee injury, knee surgery, and hand OA. RESULTS: We compared 2462 knees with pain to 1805 knees without pain. In the bivariate analysis, age, sex, BMI, injury, surgery, and hand OA were associated with at least one pain pattern. In the multivariate model, all of these variables remained significantly associated with at least one pattern. When compared to knees without pain, higher BMI, injury, and surgery were associated with all patterns. BMI had its strongest association with diffuse pain. Older age was less likely to be associated with localized pain while female sex was associated with regional pain. CONCLUSIONS: We have shown that specific OA risk factors are associated with different knee pain patterns. Better understanding of the relationship between OA risk factors and knee pain patterns may help to characterize the heterogeneous subsets of knee OA.

Donor sex and age influence the chondrogenic potential of human femoral bone marrow stem cells.

OBJECTIVE: Damaged articular cartilage does not heal well and can progress to osteoarthritis (OA). Human bone marrow stem cells (BMC) are promising cells for articular cartilage repair, yet age- and sex-related differences in their chondrogenesis have not been clearly identified. The purpose of this study is to test whether the chondrogenic potential of human femoral BMC varies based on the sex and/or age of the donor. DESIGN: BMC were isolated from 21 males (16-82 years old (y.o.)) and 20 females (20-77 y.o.) during orthopaedic procedures. Cumulative population doubling (CPD) was measured and chondrogenesis was evaluated by standard pellet culture assay in the presence or absence of transforming growth factor beta 1 (TGFbeta1). Pellet area was measured, and chondrogenic differentiation was determined by Toluidine blue and Safranin O-Fast green histological grading using the Bern score and by glycosaminoglycan (GAG) content. RESULTS: No difference in CPD was observed due to donor sex or age. The increase in pellet area with addition of TGFbeta1 and the Bern score significantly decreased with increasing donor age in male BMC, but not in female BMC. A significant reduction in GAG content per pellet was also observed with increasing donor age in male BMC. This was not observed in female BMC. CONCLUSIONS: This study showed an age-related decline in chondroid differentiation with TGFbeta1 stimulation in male BMC, but not in female BMC. Understanding the mechanisms for these differences will contribute to improved clinical use of autologous BMC for articular cartilage repair, and may lead to the development of customized age- or sex-based treatments to delay or prevent the onset of OA.

Assessing degeneration of human articular cartilage with ultra-short echo time (UTE) T2* mapping.

OBJECTIVE: To examine the sensitivity of ultra-short echo time (UTE) T(2)* mapping to collagen matrix degeneration in human articular cartilage. METHODS: Magnetic resonance imaging (MRI) UTE-T(2)* maps and standard T(2) maps were acquired on four human tibial plateau explants. Thirty-three osteochondral cores were harvested for polarized light microscopy (PLM), and composition analyses. Collagen matrix integrity was evaluated from PLM and histological images. Matrix integrity and composition was compared to standard T(2) values and UTE-T(2)* values on a spatially registered basis. RESULTS: UTE-T(2)* values varied with matrix degeneration (P=0.008) and were lower in severely degraded cartilage compared to healthy tissue (P=0.012). A trend for higher UTE-T(2)* values in healthy tissue compared to mildly degenerate tissue (P=0.051) was detected. Standard T(2) values were not found to vary with matrix degeneration (P=0.13) but tended to be higher in severely degraded cartilage compared to healthy tissue. UTE-T(2)* value variations were independent of type-II collagen and glycosaminoglycan contents. UTE-T(2)* mapping of deep cartilage, adjacent to subchondral bone, was more robust than standard T(2) mapping in this zone. CONCLUSION: UTE-T(2)* mapping of articular cartilage is sensitive to matrix degeneration and detects short-T(2) signal, particularly in deep tissue, that is not well captured by standard T(2) mapping. Correlation of UTE-T(2)* values and PLM indices supports the hypothesis that both may be sensitive to collagen microstructure. Further exploration of UTE-T(2)* mapping as a non-invasive tool to detect early articular cartilage degeneration is warranted.

Advances in Tissue Engineering Techniques for Articular Cartilage Repair.

The limited repair potential of human articular cartilage contributes to development of debilitating osteoarthritis and remains a great clinical challenge. This has led to evolution of cartilage treatment strategies from palliative to either reconstructive or reparative methods in an attempt to delay or "bridge the gap" to joint replacement. Further development of tissue engineering-based cartilage repair methods have been pursued to provide a more functional biological tissue. Currently, tissue engineering of articular cartilage has three cornerstones; a cell population capable of proliferation and differentiation into mature chondrocytes, a scaffold that can host these cells, provide a suitable environment for cellular functioning and serve as a sustained-release delivery vehicle of chondrogenic growth factors and thirdly, signaling molecules and growth factors that stimulate the cellular response and the production of a hyaline extracellular matrix (ECM). The aim of this review is to summarize advances in each of these three fields of tissue engineering with specific relevance to surgical techniques and technical notes.

The in vitro effects of bupivacaine on articular chondrocytes.

We have studied the effects of bupivacaine on human and bovine articular chondrocytes in vitro. Time-lapse confocal microscopy of human articular chondrocytes showed > 95% cellular death after exposure to 0.5% bupivacaine for 30 minutes. Human and bovine chondrocytes exposed to 0.25% bupivacaine had a time-dependent reduction in viability, with longer exposure times resulting in higher cytotoxicity. Cellular death continued even after removal of 0.25% bupivacaine. After exposure to 0.25% bupivacaine for 15 minutes, flow cytometry showed bovine chondrocyte viability to be 41% of saline control after seven days. After exposure to 0.125% bupivacaine for up to 60 minutes, the viability of both bovine and human chondrocytes was similar to that of control groups. These data show that prolonged exposure 0.5% and 0.25% bupivacaine solutions are potentially chondrotoxic.

Adeno-associated viral gene transfer of transforming growth factor-beta1 to human mesenchymal stem cells improves cartilage repair.

Bone marrow cells are routinely accessed clinically for cartilage repair. This study was performed to determine whether adeno-associated virus (AAV) effectively transduces human bone marrow-derived mesenchymal stem cells (hMSC) in vitro, whether AAV infection interferes with hMSC chondrogenesis and whether AAV-transforming growth factor-beta-1 (TGF-beta1)-transduced hMSC can improve cartilage repair in vivo. Adult hMSC were transduced with AAV-green fluorescent protein (GFP) or AAV-transforming growth factor beta1 (TGF beta1) and studied in pellet cultures. For in vivo studies, AAV-GFP and AAV-TGF-beta1-transduced hMSCs were implanted into osteochondral defects of 21 athymic rats. GFP was detected using fluorescent microscopy. Cartilage repair was assessed using gross and histological analysis at 4, 8 and 12 weeks. In pellet culture, GFP expression was visualized in situ through 21 days in vitro. In vivo GFP transgene expression was observed by in situ fluorescent surface imaging in 100% of GFP implanted defects at 2 , 67% at 8 and 17% at 12 weeks. Improved cartilage repair was observed in osteochondral defects implanted with AAV-TGF-beta1-transduced hMSC at 12 weeks (P=0.0047). These results show that AAV is a suitable vector for gene delivery to improve the cartilage repair potential of human mesenchymal stem cells.

Analysis of rabbit articular cartilage repair after chondrocyte implantation using optical coherence tomography.

OBJECTIVE: To evaluate the utility and limitations of optical coherence tomography (OCT) for immediate, high-resolution structural analysis of rabbit articular repair tissue following chondrocyte implantation without excising or sectioning the specimen. METHODS: Full thickness articular cartilage defects were created in the patellar grooves of 30 adult rabbit knee joints. Allogenic cultured chondrocytes embedded in collagen gels were implanted into the surgical defects. A periosteal patch was then sutured over the chondrocyte-collagen composites. Six animals per time point were sacrificed at 2, 4, 8, 12 and 24 weeks after surgery. The repair tissues were sequentially analysed by arthroscopic surface imaging, OCT, and histology. The resulting images were compared to determine qualitative and quantitative features of surface roughness, repair tissue integration, and micro-architecture. Statistical analysis was performed using Student's t -testing and linear regression. RESULTS: OCT was able to identify the bone and cartilage interface in normal rabbit articular cartilage and regenerated cartilage at 24 weeks post chondrocyte implantation. OCT was able to identify hypertrophy at 4 and 8 weeks, and subtle surface fibrillations at 24 weeks, comparable with histological analysis at low magnification (20x). More importantly, OCT was able to detect embedded gaps between the repair tissue and surrounding host cartilage. CONCLUSION: Close correlation was observed between OCT and histological analysis of morphological features important to the assessment of articular cartilage repair. These results demonstrate that OCT is capable of providing immediate 'optical biopsy' of the rabbit articular cartilage repair tissue without damaging the specimen, and suggest that this new technique, if integrated with an arthroscope, can potentially be used in longitudinal studies of articular cartilage repair in vivo.

Articular cartilage transplantation. Clinical results in the knee.

Between December 1983 and August 1991, 55 consecutive patients (55 knees) who underwent articular cartilage transplantation to their damaged knees were enrolled in the study. Average followup was 75 months (range, 11-147 months). Eight-two percent were younger than 45 years of age. Patients were evaluated through an 18-point scale, with 6 points each allocated to pain, range of motion, and function. An excellent knee was pain free, had full range of motion, and permitted unlimited activity. A good knee allowed full time employment and moderate activity. Eleven of 15 (73%) allografts transplanted 10 or more years ago were still good or excellent at the time of last followup. Overall, 45 of 55 (76%) knees that received the transplants were rated good or excellent. Specifically, 36 of 43 (84%) patients with unipolar transplants regained normal use of their resurfaced knee. The results after bipolar resurfacing were less encouraging, with only six of 12 (50%) knees rated good or excellent. The described technique of osteochondral shell allograft resurfacing of the knee capitalize on the different healing potentials of bone and cartilage by transplanting the viable articular cartilage organ in its entirety along with just enough of the underlying bone to allow for graft incorporation through creeping substitution. The results support the use of fresh osteochondral shell allograft transplantation for the treatment of large, full thickness articular cartilage defects to the medial or lateral femoral condyles and to the patella.

Osteochondral repair using perichondrial cells. A 1-year study in rabbits.

Articular cartilage repair remains a clinical and scientific challenge with increasing interest focused on the transplantation of chondrogenic cells. This study evaluated the repair response during a 1-year period after implantation of allogenic perichondrium cell polylactic acid composite grafts into 3.7 x 5 mm osteochondral defects drilled into the medial femoral condyles of 82 adult New Zealand White rabbits. The repair tissue was evaluated grossly, histologically, histomorphometrically, biochemically, and biomechanically at 6 weeks, 12 weeks, 6 months, and 1 year after implantation. After gross evaluation, cartilaginous material appeared to fill the defect in 70 experimental knees, for an overall repair frequency of 85%. The histomorphometric results and the histologic appearances were variable. None of the specimens were completely normal at 1 year. Only specimens with subchondral bone reformation displayed a definable cartilage appearing surface with chondrocytes surrounded by dense matrix. Subchondral bone reformation was inconsistent, reaching 50% at 1 year. Biochemically, the repair tissue matured during a 1-year period into a hyaline Type II collagen dominant tissue, whereas glycosaminoglycan content remained low at all time periods. The measured compressive properties of the repair tissue at 1 year were not significantly different from those of the contralateral knee that was not surgically treated. The treatment of osteochondral defects in the rabbit knee with allogenic perichondrium cell polylactic acid composite grafts yielded a high percentage of grossly successful repairs that showed inconsistent subchondral bone reformation. These results suggest that healthy subchondral bone is important to articular cartilage repair. They also highlight that a cartilaginous appearing tissue at gross inspection may not represent structurally normal articular cartilage. Continued multidisciplinary studies on the arthroplastic potential of rib perichondrial cells are needed before human studies, which rarely can extend beyond gross assessment of repair tissue appearance can be undertaken.

The Marshall R. Urist Young Investigator Award. Autogenous flexor tendon grafts. Biologic mechanisms for incorporation.

To examine the hypothesis that different types of dense regular connective tissue may have different repair mechanisms within the synovial space, intrasynovial and extrasynovial autogenous donor flexor tendon grafts were placed within the synovial sheaths of the medial and lateral forepaw digits of dogs. Histologic, ultrastructural, biochemical, and biomechanical analyses were done between 10 days and 6 weeks after tendon grafting. Intrasynovial tendon grafts remained viable when transferred to the synovial space and appeared to heal through an intrinsic process with preservation of the gliding surface and improved functional characteristics. Extrasynovial tendon grafts functioned as a scaffolding for the early ingrowth of new vessels and cells. Early cellular necrosis consistently was followed by the ingrowth of fibrovascular adhesions from the periphery. The formation of dense peripheral adhesions, obliterating the gliding surface of the tendon, led to diminished tendon excursion and proximal interphalangeal joint rotation.

In situ assessment of cell viability within biodegradable polylactic acid polymer matrices.

Efforts to expand treatment options for articular cartilage repair have increasingly focussed on the implantation of cell polymer constructs. Primary cells cultured from perichondrium, a chondrogenic tissue, were found to survive in vitro within a biodegradable porous polylactic acid matrix. The novel application of an in situ fluorescent double-stain protocol to cell polymer constructs was supported by increased 3H-thymidine uptake and the ability of cell seeded polylactic acid to form first passage explant cultures. This in situ viability staining technique allowed for rapid determination of cell viability and, in conjunction with confocal microscopy, assessment of cellular distribution within a biodegradable scaffold. Advantages of using this method over histological and electron microscopic analysis include in situ observation, absence of distortion in scaffold architecture due to polymer dissolution and disruption during processing, and obtaining a viability assessment within 30 min. Potential applications of this protocol as a screening tool for laboratory engineered tissues and in the evaluation of cellular injury in natural tissues are discussed.

Articular cartilage repair using allogeneic perichondrocyte-seeded biodegradable porous polylactic acid (PLA): a tissue-engineering study.

Efforts to expand treatment options for articular cartilage repair have increasingly focused on the implantation of cell-polymer constructs. The purpose of this study is to determine the suitability of porous D,D-L,L-polylactic acid as a carrier for delivering repair cells obtained from rib perichondrium into full-thickness articular cartilage defects. In vitro characterization of perichondrocyte-polylactic acid composite grafts was combined with in vivo assessment of the early articular cartilage repair in a clinically relevant model. Using a fluorescent double-stain protocol to visualize live and dead cells in situ, primary cells cultured from perichondrium were found to be capable of attaching to and surviving within a porous D,D-L,L-polylactic acid matrix. These perichondrocyte-polylactic acid composite grafts were then implanted within osteochondral defects drilled into the left medial femoral condyles of 16 adult New Zealand white rabbits. Experimental animals were sacrificed 6 weeks after implantation and the repair tissue was evaluated grossly, histologically, and biochemically. Grossly, 96% (15/16) of the experimental animals demonstrated repairs consisting of a smooth, firm neocartilage which appeared similar in color and texture to the surrounding articular surface. Matrix staining for cartilaginous protein was seen surrounding chondrocyte-like cells in the cartilage regions of the repair. Cellular alignment was found to be related to scaffold architecture. These results suggest that scaffolds composed of porous D,D-L,L-polylactic acid support the growth of cartilaginous repair tissue and are compatible with both in vitro and in vivo survival of chondrogenic cells.