Intra-articular injection of synovial mesenchymal stem cells improves cartilage repair in a mouse injury model.

Controversy remains whether articular cartilage has an endogenous stem/progenitor cell population, since its poor healing capacity after injury can lead to diseases such as osteoarthritis. In the joint environment there are mesenchymal stem/progenitor cells (MSCs) in the synovial membrane and synovial fluid that can differentiate into cartilage, but it is still under debate if these cells contribute to cartilage repair in vivo. In this study, we isolated a Sca-1 positive, chondrogenesis capable population of mouse synovial MSCs from C57BL6 and MRL/MpJ "super-healer" strains. Intra-articular injection of Sca-1 + GFP + synovial cells from C57BL6 or MRL/MpJ into C57BL6 mice following cartilage injury led to increased cartilage repair by 4 weeks after injury. GFP expression was detected in the injury site at 2 weeks, but not 4 weeks after injury. These results suggest that synovial stem/progenitor cells, regardless of strain background, have beneficial effects when injected into an injured joint. MSCs derived from MRL/MpJ mice did not promote an increased repair capacity compared to MSCs derived from non-healing C57BL6 controls; however, MRL/MpJ MSCs were observed within the defect area at the time points examined, while C57BL6 MSCs were not.

Embryonic stem cell therapy improves bone quality in a model of impaired fracture healing in the mouse; tracked temporally using in vivo micro-CT.

In the current study, we used an estrogen-deficient mouse model of osteoporosis to test the efficacy of a cell-generated bone tissue construct for bone augmentation of an impaired healing fracture. A reduction in new bone formation at the defect site was observed in ovariectomized fractures compared to the control group using repeated measures in vivo micro-computed tomography (µCT) imaging over 4 weeks. A significant increase in the bone mineral density (BMD), trabecular bone volume ratio, and trabecular number, thickness and connectivity were associated with fracture repair in the control group, whereas the fractured bones of the ovariectomized mice exhibited a loss in all of these parameters (p<0.001). In a separate group, ovariectomized fractures were treated with murine embryonic stem (ES) cell-derived osteoblasts loaded in a three-dimensional collagen I gel and recovery of the bone at the defect site was observed. A significant increase in the trabecular bone volume ratio (p<0.001) and trabecular number (p<0.01) was observed by 4 weeks in the fractures treated with cell-loaded collagen matrix compared to those treated with collagen I alone. The stem cell-derived osteoblasts were identified at the fracture site at 4 weeks post-implantation through in situ hybridization histochemistry. Although this cell tracking method was effective, the formation of an ectopic cellular nodule adjacent to the knee joints of two mice suggested that alternative in vivo cell tracking methods should be employed in order to definitively assess migration of the implanted cells. To our knowledge, this study is the first of its kind to examine the efficacy of stem cell therapy for fracture repair in an osteoporosis-related fracture model in vivo. The findings presented provide novel insight into the use of stem cell therapies for bone injuries.

Static compressive loading reduces the mRNA expression of type II and X collagen in rat growth-plate chondrocytes during postnatal growth.

The mechanisms by which chondrocytes modulate longitudinal bone growth are not well understood. This in vitro study investigated the effects of loading on the mRNA expression pattern of key molecular components of the growth-plate related to the extracellular matrix (type II and type X collagen) and the PTH-PTHrP feedback loop. Short-term static compressive loading was applied to rat proximal tibial growth-plate explants. Four age groups at specific developmental stages were investigated. The spatial variation in the mRNA expression was compared among loaded explants, their contralateral sham controls, and uncultured growth plates from normal animals. Basic cell metabolism (18S rRNA) was unaffected by load. Results indicated a narrower spatial distribution of mRNA expression of type II collagen throughout the growth plate; similarly, a narrowed distribution of expression of type X collagen was noted in the lower hypertrophic zone of the growth-plate. This suggests that mechanical compression influences chondrocytes of the hypertrophic zone to alter their expression of specific genes encoding proteins of the extracellular matrix, while PTH-PTHrP receptor mRNA, a regulatory protein, remained unaffected by loading. The effects of compression were similar at the different stages of growth, suggesting that additional factors may be involved in the clinical progression of skeletal deformities observed during growth spurts. Although this study was done in vitro and limited to static loading, it furthers our understanding of growth-plate mechanobiology as a first step toward providing a scientific rationale for treating progressive musculoskeletal deformities.

Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy.

OBJECTIVES: To detect changes in the collagen fibril network in articular cartilage in a canine experimental model of early osteoarthritis (OA) using microscopic magnetic resonance imaging (microMRI) and polarised light microscopy (PLM). METHODS: Eighteen specimens from three pairs of the medial tibia of an anterior cruciate ligament transection canine model were subjected to microMRI and PLM study 12 weeks after surgery. For each specimen, the following experiments were carried out: (a) two dimensional microMRI images of T(2) relaxation at four orientations; (b) the tangent Young's modulus; and (c) two dimensional PLM images of optical retardance and fibril angle. Disease induced changes in tissue were examined across the depth of the cartilage at a microMRI resolution of 13.7-23.1 microm. RESULTS: Several distinct changes from T(2) weighted images of cartilage in OA tibia were seen. For the specimens that were covered at least in part by the meniscus, the significant changes in microMRI included a clear shift in the depth of maximum T(2) (21-36%), a decrease in the superficial zone thickness (37-38%), and an increase in cartilage total thickness (15-27%). These microMRI changes varied topographically in the tibia surface because they were not significant in completely exposed locations in medial tibia. The microMRI results were confirmed by the PLM measurements and correlated well with the mechanical measurements. CONCLUSION: Both microMRI and PLM can detect quantitatively changes in collagen fibre architecture in early OA and resolve topographical variations in cartilage microstructure of canine tibia.

Angiogenesis in the distal femoral chondroepiphysis of the rabbit during development of the secondary centre of ossification.

In the developing chondroepiphyses of long bones, the avascular cartilaginous anlage is invaded by numerous blood vessels, through the process of angiogenesis. The objective of this study was to investigate the chronology of this vascular invasion with the spontaneous calcification of the cartilaginous epiphysis during development of the secondary ossification centre in the rabbit distal femur. The time-course of chondroepiphyseal vascular invasion was determined histologically and standardized for eight gestational and four postnatal intervals by plotting kit body mass against crown-rump length. Similarly, microcomputed tomography (micro-CT) helped to visualize calcification at those same gestational and postnatal intervals. To confirm the angiogenic nature of the avascular chondroepiphysis, such samples were assayed on the chick chorio-allantoic membrane (CAM). Neovascular outgrowths from the CAM were apparent 48 h following introduction of an 18-day (gestational) chondroepiphyseal sample. Chondroepiphyseal samples were assayed for the potent developmental angiogenic factors bFGF and VEGF, with the mRNA expression for both these mediators being confirmed using RT-PCR. As angiogenesis and calcification during chondroepiphyseal development occur in a defined tissue environment initially devoid of blood vessels and mineral, those processes provided a unique opportunity to study their progression without complication of injury-related inflammation or extant vasculature and mineral. Furthermore, the discovery of angiogenic, angiostatic or mineral-regulating mediators specific to developing connective tissue may prove useful for analysing the regulation of vascular and mineral pathogenesis in articular tissues.

Absolute concentrations of mRNA for type I and type VI collagen in the canine meniscus in normal and ACL-deficient knee joints obtained by RNase protection assay.

Relatively little is known about the cellular and molecular responses of the knee joint meniscus to joint injury, despite the functional importance of the tissue. We investigated how meniscus cells respond to joint injury in the early stages of post-traumatic osteoarthritis by characterizing the changes in matrix gene expression in menisci at 3 and 12 weeks post-surgery in dogs in which the anterior cruciate ligament (ACL) in one joint was transected and the other unoperated joint served as a control. Changes in the total RNA and DNA concentrations of the menisci were determined. Absolute concentrations of the mRNA of the COL1A1 gene of type 1 collagen, the major fibrillar collagen of the meniscus, and the COL6A3 gene of type VI collagen, a major repair molecule, were determined by quantitative ribonuclease (RNase) protection assay. The concentration of total RNA in medial and lateral menisci increased from 40 to 60 microg RNA/g wet wt in unoperated, control joints to 200-350 microg RNA/g wet wt in ACL-deficient joints. No significant changes were detected in the concentration of DNA (900-1200 microg DNA/g wet wt). Low concentrations of COL1A1 (2-3 pmol mRNA/g DNA) and COL6A3 (0.3-0.6 pmol mRNA/g DNA) mRNA transcripts were measured in normal menisci. ACL-deficiency induced a 20-38 fold increase in COL1A1 and COL6A3 mRNA concentration at 3 weeks, and an 11-19 fold increase at 12 weeks post-surgery. In general, the increase in COL1A1 and COL6A3 mRNA concentrations was greater in medial menisci than in lateral menisci. These results demonstrate that the menisci initiate a vigorous biosynthetic response to transection of the ACL.

Vascular alterations in the rabbit patellar tendon after surgical incision.

Open incision of the patellar tendon (PT) is thought to promote acute vascular responses which ultimately result in an enhanced degree of tendon repair. Such a clinical procedure is commonly applied to patients with refractory tendinitis. The objective of this study was to quantify the vascular adaptations (both anatomical and physiological) to longitudinal incision of the PT, and the resultant effects on tendon organisation. Fifty-four New Zealand White rabbits were separated into 3 experimental groups and 2 control groups. Experimental groups underwent surgical incision of the right PT, and were assessed 3 d, 10 d and 42 d following injury; normal unoperated controls were evaluated at time zero, and sham-operated controls were evaluated at 3 d to control for the effects of incising the overlying skin. Quantitative measures of PT blood supply (blood flow, microvascular volume) and geometric properties of PT substance were obtained for each PT. Histomorphology was assessed to evaluate vascular remodelling and matrix organisation in the healing PT. Longitudinal open incision surgery of the PT led to rapid increases in both blood flow and vascular volume. The incision of overlying tissues alone (sham-operated) contributed to this measurable increase, and accounted for 36% and 42% of the elevated blood flow and vascular volume respectively at the 3 d interval. In the incised PT, blood flow significantly increased by 3 d compared with both time zero and sham-operated controls, and remained significantly elevated at the 10 d interval. Similarly, vascular volume of the incised PT increased at 3 d compared both with time zero and sham-operated controls. At the 10 d interval, the increase in vascular volume was greatest in the central PT substance. By 42 d both blood flow and vascular volume of the incised tendon had diminished, with only blood flow remaining significantly different from controls. In the contralateral limb, a significant neurogenically mediated vasodilation was measured in the contralateral PTs at both early time intervals, but was not seen by the later 42 d interval. With respect to PT geometric properties in the experimental animals, a larger PT results as the tendon matrix and blood vessels remodel. PT cross-sectional area increased rapidly by 3 d to 1.3 times control values, and remained significantly elevated at 42 d postinjury. Morphological assessments demonstrated the disruption of matrix organisation by vascular and soft tissue components associated with the longitudinal incisions. Substantial changes in matrix organisation persisted at 42 d after surgery. These findings suggest that open longitudinal incision of the PT increases the vascular supply to deep tendon early after injury. These changes probably arise through both vasomotor and angiogenic activity in the tissue. Since PT blood flow and vascular volume return towards control levels after 6 wk but structural features remain disorganised, we propose that vascular remodelling is more rapid and complete than matrix remodelling after surgical incision of the PT.

The early molecular natural history of experimental osteoarthritis. I. Progressive discoordinate expression of aggrecan and type II procollagen messenger RNA in the articular cartilage of adult animals.

OBJECTIVE: To quantify changes in the chondrocyte metabolism of aggrecan core protein and type II procollagen messenger RNA (mRNA) during the early and middle phases of experimental osteoarthritis (OA) in animals. METHODS: Experimental OA was induced by transecting the cranial cruciate ligament of the stifle joint in adult animals; articular cartilage was harvested and analyzed after 4, 10, and 32 weeks. RESULTS: Northern blot analysis revealed no change in aggrecan mRNA 4 weeks after surgery compared with aggrecan mRNA in the unoperated contralateral control joints; aggrecan mRNA levels became significantly elevated by 10 and 32 weeks after surgery. In OA cartilage, type II procollagen mRNA was dramatically and progressively elevated at all times after surgery. The relative increases in type II procollagen mRNA exceeded the relative increases in aggrecan mRNA at all times after surgery, and these differences increased progressively over time. Articular chondrocytes became activated globally (total RNA increases) and specifically (mRNA increase) early after joint injury and remained activated throughout the early and middle phases of this experimental OA. CONCLUSION: The early natural history of experimental OA is characterized by a progressive imbalance in the mRNA expression of aggrecan and type II procollagen in articular chondrocytes. These results suggest that the stimuli for the transcription of these 2 genes are fundamentally different in this animal model.

Enhanced glomerular retrieval for renal biopsies.

A substantial number of glomeruli were found to become dislodged from renal biopsy cores during the biopsy procedure. These "lost" glomeruli can be retrieved and immobilized on membrane substrates; the morphology of these membrane-immobilized glomeruli is of diagnostic quality. Hence, this technique of glomerular retrieval offers an opportunity to maximize the number of glomeruli obtained at biopsy and also makes available additional material for diagnostic studies, physiology studies, and archiving.

Hindlimb loading, morphology and biochemistry of articular cartilage in the ACL-deficient cat knee.

The cat hindlimb is the best studied animal model of neuromuscular control, muscle mechanics/muscle morphology, and locomotor kinematics. Therefore, this model offers itself for intervention studies, where a musculoskeletal parameter is perturbed and the effects of this perturbation are compared with normal function. The objective of this study was to describe the effects of anterior cruciate ligament (ACL) transection in the cat knee and to correlate hindlimb loading with morphological and biochemical changes of articular cartilage. A distinct unloading of the deficient hindlimb was found when compared with the nonoperated hindlimb immediately after ACL transection, and persisted for about 16-18 weeks. Beyond about 18 weeks post-ACL transection, hindlimb loading returned to the symmetric pattern observed before surgery. In accordance with the expectations from the force-platform results, a decrease in muscle mass was found from muscles of the experimental hindlimb when compared to the mass of muscles from the contralateral hindlimb. This decrease of muscle mass was largest at 4 weeks and smallest at 35 weeks post-ACL transection. At 12 and 35 weeks post-ACL transection, cell density was increased and absolute amounts of hexuronic acid were elevated in the articular cartilage of the experimental knee compared with the corresponding values of the contralateral knee. Progressive changes of the articular cartilage towards osteoarthritis (OA) were not observed in the time frame of this experiment. The results suggest that anterior cruciate ligament transection in the cat produces initial changes in the knee that are similar to those observed previously in the dog and rabbit.

Acute surgical repair of the skier's thumb.

Complete ulnar collateral ligament tears incurred during the period fall 1977 through spring 1979 were treated under regional anesthesia by a single group of surgeons in an identical fashion as outpatients. Of 123 thumbs repaired, 69 (59%) were available for follow-up examination. There were 34 women and 35 men with an average age of 34.5 years (range, 16-61 years). The follow-up period averaged 31.6 months (range, 16-46 months). Weakness of pinch was described as none or mild in 66 (96%) and significant in only three (4%). Stiffness was described as none or mild in 66 (96%), moderate in two (3%), and severe in one (1%). Pain was described as none or mild in 68 (99%) and moderate in one (1%). Sixty-two patients (90%) preferred outpatient surgery with regional anesthesia whereas only seven (10%) did not. Forty-five (65%) of the 69 patients who required surgery had used traditional poles with straps, suggesting the pole as the causative factor. However, only 20% of noninjured skiers during the 1981-1982 season were using traditional poles with straps, and the total number of complete ulnar collateral ligament repairs did not decrease with the same number of skiers. Therefore, the change in pole design has not decreased the incidence of total ulnar collateral ligament tears. The results of acute surgical repair as an outpatient with regional anesthesia are excellent; 96% of the patients were pleased with the results. If the lesion is overlooked, the results of secondary construction are not nearly as good.