Objective-The differentiation of mesenchymal stem cells (MSCs) into chondrocytes provides an attractive basis for the repair and regeneration of articular cartilage. Under clinical conditions, chondrogenesis will often need to occur in the presence of inflammatory mediators produced in response to injury or disease. Here we examine the effect of two important inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), on the chondrogenic behavior of human MSCs.Methods-Aggregate cultures of MSCs recovered from the femoral intermedullary canal were used. Chondrogenesis was assessed by the expression of relevant transcripts by quantitative RT-PCR and examination of aggregates by histology and immunohistochemistry. The possible involvement of NF-κB in mediating the effects of IL-1β was examined by delivering a luciferase reporter construct and a dominant negative inhibitor of NF-κB (srIκB), with adenovirus vectors.Results-Both IL-1β and TNF-α inhibited chondrogenesis in a dose-dependent manner. This was associated with a marked activation of NF-κB. Delivery of srIκB abrogated the activation of NF-κB and rescued the chondrogenic response. Although expression of type X collagen followed this pattern, other markers of hypertrophic differentiation responded differently. Matrix metalloproteinase-13 was induced by IL-1β in a NF-κB dependent fashion. Alkaline phosphatase activity, in contrast, was inhibited by IL-1β regardless of srIκB delivery.Conclusions-Cell-based repair of lesions in articular cartilage will be compromised in inflamed joints. Strategies for enabling repair under these conditions include the use of specific antagonists of individual pyrogens, such as IL-1 and TNF, or the targeting of important intracellular mediators, such as NF-κB. There are two general strategies to harnessing MSCs for this purpose. In a tissue engineering approach, MSCs are recovered from the patient and used to generate a graft that is subsequently implanted into the site of cartilage damage (4). Although the graft can be developed in a bioreactor into mature cartilage, there is increasing interest in grafting immature tissue, allowing chondrogenesis to occur in situ. The second strategy, which is already in wide clinical use, supplies MSCs to the defect by penetrating the subchondral bone, thereby allowing marrow to enter the lesion. Various related surgical techniques, including microfracture and subchondral drilling, are used for this purpose. These procedures have the convenience of being performed arthroscopically in large joints (1).Repair strategies that rely on the in situ differentiation of MSCs are attractive, but in many instances require chondrogenesis to take place within an inflamed environment. Intraarticular inflammation may result from disease, such as arthritis, or trauma, including the iatrogenic trauma of the cartilage repair surgery itself. Because interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are major mediators of local inflammatory processes in joints, the present ...

When ruptured, the anterior cruciate ligament (ACL) of the human knee has limited regenerative potential. However, the goal of this report was to show that the cells that migrate out of the human ACL constitute a rich population of progenitor cells and we hypothesize that they display mesenchymal stem cell (MSC) characteristics when compared with adherent cells derived from bone marrow or collagenase digests from ACL. We show that ACL outgrowth cells are adherent, fibroblastic cells with a surface immunophenotype strongly positive for cluster of differentiation (CD)29, CD44, CD49c, CD73, CD90, CD97, CD105, CD146, and CD166, weakly positive for CD106 and CD14, but negative for CD11c, CD31, CD34, CD40, CD45, CD53, CD74, CD133, CD144, and CD163. Staining for STRO-1 was seen by immunohistochemistry but not flow cytometry. Under suitable culture conditions, the ACL outgrowth-derived MSCs differentiated into chondrocytes, osteoblasts, and adipocytes and showed capacity to self-renew in an in vitro assay of ligamentogenesis. MSCs derived from collagenase digests of ACL tissue and human bone marrow were analyzed in parallel and displayed similar, but not identical, properties. In situ staining of the ACL suggests that the MSCs reside both aligned with the collagenous matrix of the ligament and adjacent to small blood vessels. We conclude that the cells that emigrate from damaged ACLs are MSCs and that they have the potential to provide the basis for a superior, biological repair of this ligament.

Intra-articular (i.a.) drug delivery for local treatment of osteoarthritis remains inadequate due to rapid clearance by the vasculature or lymphatics. Local therapy targeting articular cartilage is further complicated by its dense meshwork of collagen and negatively charged proteoglycans, which can prevent even nano-sized solutes from entering. In a previous in vitro study, we showed that Avidin, due to its size (7 nm diameter) and high positive charge (pI 10.5), penetrated the full thickness of bovine cartilage and was retained for 15 days. With the goal of using Avidin as a nano-carrier for cartilage drug delivery, we investigated its transport properties within rat knee joints. Avidin penetrated the full thickness of articular cartilage within 6 h, with a half-life of 29 h, and stayed inside the joint for 7 days after i.a. injection. The highest concentration of Avidin was found in cartilage, the least in patellar tendon and none in the femoral bone; in contrast, negligible Neutravidin (neutral counterpart of Avidin) was present in cartilage after 24 h. A positive correlation between tissue sGAG content and Avidin uptake (R 2 ¼ 0.83) confirmed the effects of electrostatic interactions. Avidin doses up to at least 1 mM did not affect bovine cartilage explant cell viability, matrix catabolism or biosynthesis. Keywords: Avidin; intra-articular drug delivery; rat; glycosaminoglycans; cartilage Osteoarthritis (OA) affects individual joints, necessitating localized therapy. 1,2 Intra-articular (i.a.) injections allow for local and targeted delivery of drugs into the joint space, thereby reducing systemic toxicity. However, i.a. therapy often remains inadequate due to rapid clearance of drugs from the joint space; small molecules exit via the vasculature while larger macromolecules (e.g., hyaluronan) are cleared by the lymphatic system. 2,3 Mean half-lives of NSAIDs in the synovial fluid have been reported to be 1-4 h. 4,5 Solutes in synovial fluid with sizes similar to plasma proteins (albumin $67 kDa, globulin $150 kDa, fibrinogen $340 kDa) have equal permeability through the lymphatics. 2 Their clearance, however, is dependent on the rate of synovial fluid turnover and solute diffusivity; the latter is a function of solute size and molecular weight, viscosity of synovial fluid and temperature. For example, intraarticular half-lives in normal rabbit knee joints have been reported to range from 0.23 h for Acridine Blue (370 Da) to 1.23 h for Albumin (67 kDa) and 26.3 h for Hyaluronan (300 kDa). 2,3 Intra-articular injection of drug-encapsulating particles can increase half-lives of therapeutic drugs in the synovial fluid. 1,6,7 However, therapeutic efficacy depends on the ability of the drugs (or particle-bound drugs) to penetrate into specific target tissues and to be retained by those tissues over time. Entry of macromolecules into cartilage is hindered by its dense extracellular matrix (ECM) of collagen fibrils and aggrecan proteoglycans containing highly negatively charged glycosaminoglycan (GAG) chai...

Facilitated endogenous repair is a novel approach to tissue engineering that avoids the ex vivo culture of autologous cells and the need for manufactured scaffolds, while minimizing the number and invasiveness of associated clinical procedures. The strategy relies on harnessing the intrinsic regenerative potential of endogenous tissues using molecular stimuli, such as gene transfer, to initiate reparative processes in situ. In the simplest example, direct percutaneous injection of an osteogenic vector is used to stimulate bone healing. If necessary, additional progenitor cells and space-filling scaffolds can be provided by autologous bone marrow, muscle, fat, and perhaps other tissues. These can be harvested, processed, and reimplanted by simple, expedited, intraoperative procedures. Examples of repair of experimental osseous and osteochondral lesions in laboratory animals are described. If successful, these strategies will provide methods for tissue regeneration that are not only effective but also inexpensive, safe, and clinically expeditious. Although orthopaedic examples are given here, the technology should be more generally applicable.

We report a novel technology for the rapid healing of large osseous and chondral defects, based upon the genetic modification of autologous skeletal muscle and fat grafts. These tissues were selected because they not only possess mesenchymal progenitor cells and scaffolding properties, but also can be biopsied, genetically modified and returned to the patient in a single operative session. First generation adenovirus vector carrying cDNA encoding human bone morphogenetic protein-2 (Ad.BMP-2) was used for gene transfer to biopsies of muscle and fat. To assess bone healing, the genetically modified ("gene activated") tissues were implanted into 5mm-long critical size, mid-diaphyseal, stabilized defects in the femora of Fischer rats. Unlike control defects, those receiving gene-activated muscle underwent rapid healing, with evidence of radiologic bridging as early as 10 days after implantation and restoration of full mechanical strength by 8 weeks. Histologic analysis suggests that the grafts rapidly differentiated into cartilage, followed by efficient endochondral ossification. Fluorescence in situ hybridization detection of Y-chromosomes following the transfer of male donor muscle into female rats demonstrated that at least some of the osteoblasts of the healed bone were derived from donor muscle. Gene activated fat also healed critical sized defects, but less quickly than muscle and with more variability. Anti-adenovirus antibodies were not detected. Pilot studies in a rabbit osteochondral defect model demonstrated the promise of this technology for healing cartilage defects. Further development of these methods should provide ways to heal bone and cartilage more expeditiously, and at lower cost, than is presently possible.

Dexamethasone is capable of directing osteoblastic differentiation of bone marrow stromal cells (BMSCs) in vitro, but its effects are not lineage-specific, and sustained exposure has been shown to down-regulate collagen synthesis and induce maturation of an adipocyte subpopulation within BMSC cultures. Such side effects might be reduced if dexamethasone is applied in a regimented manner, but the discrete steps in osteoblastic maturation that are stimulated by dexamethasone are not known. To examine this, dexamethasone was added to medium to initiate differentiation of rat BMSCs cultures and then removed after a varying number of days. Cell layers were analyzed for cell number, rate of collagen synthesis, expression of osteocalcin (OC), bone sialoprotein (BSP) and lipoprotein lipase (LpL), and matrix mineralization. Withdrawal of dexamethasone at 3 and 10 days was found to enhance cell number relative to continuous exposure, but did not affect to decrease collagen synthesis slightly. Late markers of osteoblastic differentiation, BSP expression and matrix mineralization, were also sensitive to dexamethasone and increased systematically with exposure while LpL systematically decreased. These results indicate that dexamethasone acts at both early and late stages to direct proliferative osteoprogenitor cells toward terminal maturation.

Intramedullary nailing preceded by canal reaming is the current standard of treatment for long-bone fractures requiring stabilization. However, conventional reaming methods can elevate intramedullary temperature and pressure, potentially resulting in necrotic bone, systemic embolism, and pulmonary complications. To address this problem, a reamer irrigator aspirator (RIA) has been developed that combines irrigation and suction for reduced-pressure reaming with temperature modulation. Osseous particles aspirated by the RIA can be recovered by filtration for use as an autograft, but the flow-through is typically discarded. The purpose of this study was to assess whether this discarded filtrate has osteogenic properties that could be used to enhance the total repair potential of aspirate. RIA aspirate was collected from five patients (ages 71–78) undergoing hip hemiarthroplasty. Osseous particles were removed using an open-pore filter, and the resulting filtrate (230 ± 200 mL) was processed by Ficoll-gradient centrifugation to isolate mononuclear cells (6.2 ± 5.2 × 106 cells/mL). The aqueous supernatant contained FGF-2, IGF-I, and latent TGF-β1, but BMP-2 was below the limit of detection. The cell fraction included culture plastic-adherent, fibroblastic cells that displayed a surface marker profile indicative of mesenchymal stem cells and that could be induced along the osteogenic, adipogenic, and chondrogenic lineages in vitro. When compared to outgrowth cells from the culture of osseous particles, filtrate cells were more sensitive to seeding density during osteogenic culture but had similar capacity for chondrogenesis. These results suggest using RIA aspirate to develop improved, clinically expeditious, cost-effective technologies for accelerating the healing of bone and other musculoskeletal tissues.

The aim of our study was to evaluate the histological and biomechanical effects of BMP-12 gene transfer on the healing of rat Achilles tendons using a new approach employing a genetically modified muscle flap. Biopsies of autologous skeletal muscle were transduced with a type-five, first-generation adenovirus carrying the human BMP-12 cDNA (Ad.BMP-12) and surgically implanted around experimentally transected Achilles tendons in a rat model. The effect of gene transfer on healing was evaluated by mechanical and histological testing after 1, 2, 4 and 8 weeks. One week after surgery, the maximum failure load of the healing tendons was significantly increased in the BMP-12 group, compared with the controls, and the tendon stiffness was significantly higher at 1, 2 and 4 weeks. Moreover, the size of the rupture callus was increased in the presence of BMP-12 and there was evidence of accelerated remodeling of the lesion in response to BMP-12. Histological examination showed a much more organized and homogeneous pattern of collagen fibers at all time points in lesions treated with the BMP-12 cDNA muscle graft. Both single fibrils and the collagen fibers had a greater diameter, with a higher degree of collagen crimp than the collagen of the control groups. This was confirmed by sirius red staining in conjunction with polarized light microscopy, which showed a higher shift of small yellow-green fibers to strong yelloworange fibers after 2, 4 and 8 weeks in the presence of BMP-12 cDNA. There was also an earlier shift from fibroblasts to fibrocytes within the healing tendon, with less fat cells present in the tendons of the BMP-12 group compared with the controls. Treatment with BMP-12 cDNA-transduced muscle grafts thus produced a promising acceleration and improvement of tendon healing, particularly influencing early tissue regeneration, leading to quicker recovery and improved biomechanical properties of the Achilles tendon. Further development of this approach could have clinical applications.