John Wardale

Exploring the Application of Stem Cells in Tendon Repair and Regeneration

PURPOSE To conduct a systematic review of the current evidence for the effects of stem cells on tendon healing in preclinical studies and human studies. METHODS A systematic search of the PubMed, CINAHL (Cumulative Index to Nursing and Allied Health Literature), Cochrane, and Embase databases was performed for stem cells and tendons with their associated terminology. Data validity was assessed, and data were collected on the outcomes of trials. RESULTS A total of 27 preclinical studies and 5 clinical studies met the inclusion criteria. Preclinical studies have shown that stem cells are able to survive and differentiate into tendon cells when placed into a new tendon environment, leading to regeneration and biomechanical benefit to the tendon. Studies have been reported showing that stem cell therapy can be enhanced by molecular signaling adjunct, mechanical stimulation of cells, and the use of augmentation delivery devices. Studies have also shown alternatives to the standard method of bone marrow-derived mesenchymal stem cell therapy. Of the 5 human studies, only 1 was a randomized controlled trial, which showed that skin-derived tendon cells had a greater clinical benefit than autologous plasma. One cohort study showed the benefit of stem cells in rotator cuff tears and another in lateral epicondylitis. Two of the human studies showed how stem cells were successfully extracted from the humerus and, when tagged with insulin, became tendon cells. CONCLUSIONS The current evidence shows that stem cells can have a positive effect on tendon healing. This is most likely because stem cells have regeneration potential, producing tissue that is similar to the preinjury state, but the results can be variable. The use of adjuncts such as molecular signaling, mechanical stimulation, and augmentation devices can potentially enhance stem cell therapy. Initial clinical trials are promising, with adjuncts for stem cell therapy in development.

Effects of lactic acid and glycolic acid on human osteoblasts: a way to understand PLGA involvement in PLGA/calcium phosphate composite failure.

The use of degradable composite materials in orthopedics remains a field of intense research due to their ability to support new bone formation and degrade in a controlled manner, broadening their use for orthopedic applications. Poly (lactide‐co‐glycolide) acid (PLGA), a degradable biopolymer, is now a popular material for different orthopedic applications and is proposed for use in tissue engineering scaffolds either alone or combined with bioactive ceramics. Interference screws composed of calcium phosphates and PLGA are readily available in the market. However, some reports highlight problems of screw migration or aseptic cyst formation following screw degradation. In order to understand these phenomena and to help to improve implant formulation, we have evaluated the effects of PLGA degradation products: lactic acid and glycolic acid on human osteoblasts in vitro. Cell proliferation, differentiation, and matrix mineralization, important for bone healing were studied. It was found that the toxicity of polymer degradation products under buffering conditions was limited to high concentrations. However, non‐toxic concentrations led to a decrease in cell proliferation, rapid cell differentiation, and mineralization failure. Calcium, whilst stimulating cell proliferation was not able to overcome the negative effects of high concentrations of lactic and glycolic acids on osteoblasts. These effects help to explain recently reported clinical failures of calcium phosphate/PLGA composites, but further in vitro analyses are needed to mimic the dynamic situation which occurs in the body by, for example, culture of osteoblasts with materials that have been pre‐degraded to different extents and thus be able to relate these findings to the degradation studies that have been performed previously.

The role of platelet rich plasma in musculoskeletal science.

The idea of using platelet rich plasma (PRP) in medicine has been around since the 1970s. It is only more recently that its use has been employed in the area of musculoskeletal science. Platelet rich plasma in this area has received much media attention being used by many celebrity sports athletes for musculoskeletal injuries. Therefore it is important for the musculoskeletal practitioner to be aware of the concepts surrounding its use and application. In this article we cover what platelet rich plasma is, how it is prepared and administered, its potential clinical application, and what the current literature discusses in the various areas of musculoskeletal science.

Peripheral Blood Mononuclear Cells Enhance Cartilage Repair in in vivo Osteochondral Defect Model.

This study characterized peripheral blood mononuclear cells (PBMC) in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC’s were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC) of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1) MSC alone; (2) MSCs and PBMCs at a ratio of 20:1; (3) MSCs and PBMC at a ratio of 2:1 and (4) PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006). However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%). This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007), BMP6 10.7-fold (p = 0.0004), GDF5 2.0-fold (p = 0.002) and COL1 5.0-fold (p = 0.046). The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key effect on the derivation of a novel adherent cell population with an MSC-like phenotype. This study presents a novel and easily attainable point-of-care cell therapy with PBMCs to treat osteochondral defects in the knee avoiding any cell manipulations outside the surgical room.

Ligands for retinoic acid receptors are elevated in osteoarthritis and may contribute to pathologic processes in the osteoarthritic joint.

OBJECTIVE Vitamin A derivatives, including all-trans-retinoic acid (ATRA), have a well-established role during skeletal development and limb formation and have been shown to have profound effects on chondrocyte phenotype. The aim of this study was to elucidate the effects of retinoids and components of the retinoid metabolic pathway on chondrocyte phenotype in the tibiofemoral joints of patients with osteoarthritis (OA), to show that the retinoids can have multiple effects relevant to the OA disease process. METHODS Human explant tissue and a chondrocyte-like cell line were treated with ATRA, and the responses of 4 key markers of chondrocyte phenotype were analyzed. In addition, the effects of ATRA on a number of novel genes associated with OA were assessed using a low-density microarray containing 80 disease marker genes. RESULTS Vitamin A metabolite levels were elevated in synovial fluid, serum, and cartilage from patients with OA. Expression profiling of a retinoic acid receptor alpha coactivator protein, P/CAF, demonstrated elevated expression in patients with OA, suggesting the potential for increased signaling via the retinoid receptors in the disease. ATRA increased the levels of matrix metalloproteinase 13 and aggrecanase activity in human cartilage explants and in a human chondrocyte cell line. Furthermore, ATRA altered the expression of a wide range of relevant genes, including the types I, II, IX, and XI collagen genes, toward a nonchondrogenic and OA-like phenotype. CONCLUSION These results suggest that retinoid signaling could have a central role in OA, and that components of the pathway may provide potential disease biomarkers or targets for therapeutic intervention.

Hypoxia Modulates the Phenotype of Osteoblasts Isolated From Knee Osteoarthritis Patients, Leading to Undermineralized Bone Nodule Formation

To investigate the role of hypoxia in the pathology of osteoarthritic (OA) bone by exploring its effect on the phenotype of isolated primary osteoblasts from patients with knee OA.

Peripheral blood derived mononuclear cells enhance the migration and chondrogenic differentiation of multipotent mesenchymal stromal cells.

A major challenge in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into damaged areas and strategies to promote this should be developed. The aim of this study was to evaluate the effect of peripheral blood derived mononuclear cell (PBMC) stimulation on mesenchymal stromal cells (MSCs) derived from the infrapatellar fat pad of human OA knee. Cell migration was measured using an xCELLigence electronic migration chamber system in combination with scratch assays. Gene expression was quantified with stem cell PCR arrays and validated using quantitative real-time PCR (rtPCR). In both migration assays PBMCs increased MSC migration by comparison to control. In scratch assay the wound closure was 55% higher after 3 hours in the PBMC stimulated test group (P = 0.002), migration rate was 9 times faster (P = 0.008), and total MSC migration was 25 times higher after 24 hours (P = 0.014). Analysis of MSCs by PCR array demonstrated that PBMCs induced the upregulation of genes associated with chondrogenic differentiation over 15-fold. In conclusion, PBMCs increase both MSC migration and differentiation suggesting that they are an ideal candidate for inclusion in regenerative medicine therapies aimed at cartilage repair.

Effect of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide concentrations on the mechanical and biological characteristics of cross-linked collagen fibres for tendon repair

Reconstituted type I collagen fibres have received considerable interest as tendon implant materials due to their chemical and structural similarity to the native tissue. Fibres produced through a semi-continuous extrusion process were cross-linked with different concentrations of the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS). Tensile properties of the fibres were considered, along with imaging of both surface structure and fibrillar alignment. Resistance of the fibres to bacterial collagenase was investigated and fibre sections seeded with human tendon cells for biological characterization, including cell adhesion and proliferation. The work clearly demonstrated that whilst the concentration of EDC and NHS had no significant effect on the mechanics, a higher concentration was associated with higher collagenase resistance, but also provided a less attractive surface for cell adhesion and proliferation. A lower cross-linking concentration offered a more biocompatible material without reduction in mechanics and with a potentially more optimal degradability.

Release of growth factors from a reinforced collagen GAG matrix supplemented with platelet rich plasma: Influence on cultured human meniscal cells

Damage to meniscal cartilage has been strongly linked to accelerated articular wear and consequently to osteoarthritis. Damage might be ameliorated by delivery of growth factors from platelet rich plasma (PRP) via a fiber reinforced collagen matrix designed for meniscal repair. PRP composition, release of growth factors, and influence on meniscal cell growth and gene expression were investigated. PRP was prepared using Harvest Smartprep (HS‐PRP), Cascade Fibrinet (CF‐PRP), and a simple centrifuge protocol (DC‐PRP) from four donors each. CF‐PRP had the highest ratio of platelets, with very few other blood cell types. HS‐PRP had the highest total number of platelets but also contained high levels of red and white blood cells. Absorbed to collagen matrices HS‐PRP released the highest levels of TGF‐β1 and PDGF‐AB with DC‐PRP the most IGF‐1. Cumulative release from collagen matrix was 48 ng/cm3 IGF‐1, 96 ng/cm3 TGF‐β1, and 9.6 ng/cm3 PDGF‐AB. Collagen matrix with PRP was able to increase meniscal cell number above peripheral whole blood and up‐regulated gene expression of Aggrecan, Collagen type I (α1), and Elastin (3.3 ± 0.8‐fold, 2.9 ± 0.6‐fold, 4.0 ± 1.4‐fold, respectively). Demonstrating that PRP combined with fiber reinforced collagen matrix could influence meniscal cells and might be of use for treating meniscal defects.

Peripheral blood derived mononuclear cells enhance osteoarthritic human chondrocyte migration.

IntroductionA major problem in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into defects. Cartilage is essentially avascular and therefore its healing is not considered to involve mononuclear cells. Peripheral blood derived mononuclear cells (PBMC) offer a readily available autologous cell source for clinical use and therefore this study was designed to evaluate the effects of PBMCs on chondrocytes and cartilage.MethodsHuman primary chondrocytes and cartilage tissue explants were taken from patients undergoing total knee replacement (n = 17). Peripheral blood samples were obtained from healthy volunteers (n = 12) and mononuclear cells were isolated by density-gradient centrifugation. Cell migration and chemokinetic potential were measured using a scratch assay, xCELLigence and CyQuant assay. PCR array and quantitative PCR was used to evaluate mRNA expression of 87 cell motility and/or chondrogenic genes.ResultsThe chondrocyte migration rate was 2.6 times higher at 3 hour time point (p < 0.0001) and total number of migrating chondrocytes was 9.7 times higher (p < 0.0001) after three day indirect PBMC stimulus and 8.2 times higher (p < 0.0001) after three day direct co-culture with PBMCs. A cartilage explant model confirmed that PBMCs also exert a chemokinetic role on ex vivo tissue. PBMC stimulation was found to significantly upregulate the mRNA levels of 2 chondrogenic genes; collagen type II (COL2A1 600–fold, p < 0.0001) and SRY box 9 (SOX9 30–fold, p < 0.0001) and the mRNA levels of 7 genes central in cell motility and migration were differentially regulated by 24h PBMC stimulation.ConclusionThe results support the concept that PBMC treatment enhances chondrocyte migration without suppressing the chondrogenic phenotype possibly via mechanistic pathways involving MMP9 and IGF1. In the future, peripheral blood mononuclear cells could be used as an autologous point-ofcare treatment to attract native chondrocytes from the diseased tissue to aid in cartilage repair.