Native multipotential stromal cell colonization and graft expander potential of a bovine natural bone scaffold

Graft expanders are bone scaffolds used, in combination with autografts, to fill large bone defects in trauma surgery. This study investigates the graft expander potential of a natural bone substitute Orthoss® by studying its ability to support attachment, growth and osteogenic differentiation of neighboring multipotential stromal cells (MSCs). Material consisting of bone marrow (BM) aspirate and reamer‐irrigator‐aspirator (RIA)‐harvested autograft bone was co‐cultured with commercially available Orthoss® granules. Native MSCs attached to Orthoss® were expanded and phenotypically characterized. MSCs egress from neighboring cancelous bone was assessed in 3D Matrigel co‐cultures. MSC differentiation was evaluated using scanning electron microscopy and measuring alkaline phosphatase (ALP) activity per cell. CD45⁺ hematopoietic lineage cells and highly proliferative CD90⁺CD73⁺CD105⁺ MSCs preferentially colonized Orthoss® granules, over RIA bone chips. MSC colonization was followed by their intrinsic osteogenic differentiation, assessed as mineral deposition and gradual rise in ALP activity, even in the absence of osteogenic stimuli. When in contact with mixed cell populations and RIA chips, Orthoss® granules support the attachment, growth and osteogenic differentiation of neighboring MSCs. Therefore, natural bone substitutes similar to Orthoss® can be used as void fillers and graft expanders for repairing large bone defects in conjunction with autologous BM aspirates and autografts. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1950–1958, 2013     

Stromal cell‐derived factor‐1 and monocyte chemotactic protein‐3 improve recruitment of osteogenic cells into sites of musculoskeletal repair

Homing of osteogenic cells through the systemic circulation represents an alternative to traditional orthopedic tissue engineering approaches that focus on local cell populations. We hypothesize that expression of the chemokine, stromal cell‐derived factor‐1 (SDF‐1) or monocyte chemotactic protein‐3 (MCP‐3) may enhance homing of osteogenic cells into sites of fracture repair, as both have demonstrated promise in recruitment of marrow stromal cells (MSCs). This hypothesis was tested by transplantation of culture expanded MSCs expressing these factors adjacent to a fracture site on a collagen scaffold. One green fluorescent protein positive (GFP⁺) and one wild‐type mouse were surgically conjoined as parabiots at 7–8 weeks of age. Fibular osteotomy was performed 4 weeks after parabiosis on the hind limb of the wild‐type mouse. Mice were randomly allocated to receive one of the following five treatments: control (no scaffold), empty scaffold (no cells), or scaffold containing MSCs, scaffold containing MSCs expressing SDF‐1, or scaffold containing MSCs expressing MCP‐3. Fracture callus was harvested 2 weeks after injury, and analyzed with confocal microscopy and cell‐counting software. When compared to fracture callus treated with nontransfected MSCs, the fracture callus of mice treated with both SDF‐1 and MCP‐3 secreting MSCs demonstrated a significant increase in the number of both GFP⁺ cells (p = 0.0003, p = 0.02) and GFP⁺/AP⁺ cells (p = 0.0005, p = 0.01). These data suggest that homing of osteogenic cells from systemic circulation participate in fracture repair and that homing pathways might be modulated to enhance the contribution of circulating progenitors at the site of skeletal injury. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1064–1069, 2011     

Low‐dose BMP‐2 and MSC dual delivery onto coral scaffold for critical‐size bone defect regeneration in sheep

Tissue‐engineered constructs (TECs) combining resorbable calcium‐based scaffolds and mesenchymal stem cells (MSCs) have the capability to regenerate large bone defects. Inconsistent results have, however, been observed, with a lack of osteoinductivity as a possible cause of failure. This study aimed to evaluate the impact of the addition of low‐dose bone morphogenetic protein‐2 (BMP‐2) to MSC‐coral‐TECs on the healing of clinically relevant segmental bone defects in sheep. Coral granules were either seeded with autologous MSCs (bone marrow‐derived) or loaded with BMP‐2. A 25‐mm‐long metatarsal bone defect was created and stabilized with a plate in 18 sheep. Defects were filled with one of the following TECs: (i) BMP (n = 5); (ii) MSC (n = 7); or (iii) MSC‐BMP (n = 6). Radiographic follow‐up was performed until animal sacrifice at 4 months. Bone formation and scaffold resorption were assessed by micro‐CT and histological analysis. Bone union with nearly complete scaffold resorption was observed in 1/5, 2/7, and 3/6 animals, when BMP‐, MSC‐, and MSC‐BMP‐TECs were implanted, respectively. The amount of newly formed bone was not statistically different between groups: 1074 mm³ [970–2478 mm³], 1155 mm³ [970–2595 mm³], and 2343 mm³ [931–3276 mm³] for BMP‐, MSC‐, and MSC‐BMP‐TECs, respectively. Increased scaffold resorption rate using BMP‐TECs was the only potential side effect observed. In conclusion, although the dual delivery of MSCs and BMP‐2 onto a coral scaffold further increased bone formation and bone union when compared to single treatment, results were non‐significant. Only 50% of the defects healed, demonstrating the need for further refinement of this strategy before clinical use. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2637–2645, 2017.

     

Selective laser melting‐produced porous titanium scaffolds regenerate bone in critical size cortical bone defects

Porous titanium scaffolds have good mechanical properties that make them an interesting bone substitute material for large bone defects. These scaffolds can be produced with selective laser melting, which has the advantage of tailoring the structure's architecture. Reducing the strut size reduces the stiffness of the structure and may have a positive effect on bone formation. Two scaffolds with struts of 120‐µm (titanium‐120) or 230‐µm (titanium‐230) were studied in a load‐bearing critical femoral bone defect in rats. The defect was stabilized with an internal plate and treated with titanium‐120, titanium‐230, or left empty. In vivo micro‐CT scans at 4, 8, and 12 weeks showed more bone in the defects treated with scaffolds. Finally, 18.4 ± 7.1 mm³ (titanium‐120, p = 0.015) and 18.7 ± 8.0 mm³ (titanium‐230, p = 0.012) of bone was formed in those defects, significantly more than in the empty defects (5.8 ± 5.1 mm³). Bending tests on the excised femurs after 12 weeks showed that the fusion strength reached 62% (titanium‐120) and 45% (titanium‐230) of the intact contralateral femurs, but there was no significant difference between the two scaffolds. This study showed that in addition to adequate mechanical support, porous titanium scaffolds facilitate bone formation, which results in high mechanical integrity of the treated large bone defects. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 792–799, 2013     

Comparative Evaluation of the Book‐Type Acellular Bone Scaffold and Fibrocartilage Scaffold for Bone‐Tendon Healing

Bone‐tendon (B‐T) healing is a clinical challenge due to its limited regeneration capability. Fibrocartilage regeneration and bone formation at the healing site are two critical factors for B‐T healing. Promoting fibrocartilage regeneration and bone formation by tissue‐engineering may be a promising treatment strategy. In this study, we innovatively fabricated two kinds of acellular scaffolds from bone or fibrocartilage tissues, namely the book‐type the acellular bone scaffold (BABS) and the book‐type acellular fibrocartilage scaffold (BAFS). Histologically, the two scaffolds well preserved the native extracellular matrix (ECM) structure without cellular components. In vitro studies showed BABS is superior in osteogenic inducibility, while BAFS has good chondrogenic inducibility. To comparatively investigate the efficacy on B‐T healing, the BABS or BAFS were, respectively, implanted into a rabbit partial patellectomy model. Macroscopically, a regenerated bone‐tendon insertion (BTI) was bridging the residual patella and patellar‐tendon with no signs of infection and osteoarthritis. Radiologically, more new bone was formed at the healing interface in the BABS group as compared with the BAFS or control (CTL) groups (p < 0.05). Histologically, at postoperative week 16, histological scores were significantly better for regenerated fibrocartilage in the BAFS group or BABS group compared with the CTL group, but the BAFS group showed a significantly larger score than the BABS groups (p < 0.05). Biomechanical evaluation indicated a higher failure load and stiffness were shown in the BAFS group than those in the BABS or CTL groups at week 16 (p < 0.05). This study indicated that the BAFS is a more promising scaffold for B‐T healing in comparison with the BABS. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1709–1722, 2019     

Repair of Critical‐Sized Long Bone Defects Using Dipyridamole‐Augmented 3D‐Printed Bioactive Ceramic Scaffolds

There are over two million long bone defects treated in the United States annually, of which ~5% will not heal without significant surgical intervention. While autogenous grafting is the standard of care in simple defects, a customized scaffold for large defects in unlimited quantities is not available. Recently, a three‐dimensionally (3D)‐printed bioactive ceramic (3DPBC) scaffold has been successfully utilized in the of repair critical‐sized (CSD) long bone defects in vivo. In this study, 3DPBC scaffolds were augmented with dipyridamole (DIPY), an adenosine A2A receptor (A_2AR) indirect agonist, because of its known effect to enhance bone formation. CSD full thickness segmental defects (~11 mm × full thickness) defects were created in the radial diaphysis in New Zealand white rabbits (n = 24). A customized 3DPBC scaffold composed of β‐tricalcium phosphate was placed into the defect site. Groups included scaffolds that were collagen‐coated (COLL), or immersed in 10, 100, or 1,000 μM DIPY solution. Animals were euthanized 8 weeks post‐operatively and the radii/ulna‐scaffold complex retrieved en bloc, for micro‐CT, histological, and mechanical analysis. Bone growth was assessed exclusively within scaffold pores and evaluated by microCT and advanced reconstruction software. Biomechanical properties were evaluated utilizing nanoindentation to assess the newly regenerated bone for elastic modulus (E) and hardness (H). MicroCT reconstructions illustrated bone in‐growth throughout the scaffold, with an increase in bone volume dependent on the DIPY dosage. The histological evaluation did not indicate any adverse immune response while revealing progressive remodeling of bone. These customized biologic 3DPBC scaffolds have the potential of repairing and regenerating bone. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2499–2507, 2019     

[18F]‐Sodium Fluoride PET/MR Imaging for Bone–Cartilage Interactions in Hip Osteoarthritis: A Feasibility Study

This study characterized the distribution of [¹⁸F]‐sodium fluoride (NaF) uptake and blood flow in the femur and acetabulum in hip osteoarthritis (OA) patients to find associations between bone remodeling and cartilage composition in the presence of morphological abnormalities using simultaneous positron emission tomography and magnetic resonance imaging (PET/MR), quantitative magnetic resonance imaging (MRI) and femur shape modeling. Ten patients underwent a [¹⁸F]‐NaF PET/MR dynamic scan of the hip simultaneously with: (i) fast spin‐echo CUBE for morphology grading and (ii) T_1ρ/T₂ magnetization‐prepared angle‐modulated partitioned k‐space spoiled gradient echo snapshots for cartilage, bone segmentation, bone shape modeling, and T_1ρ/T₂ quantification. The standardized uptake values (SUVs) and Patlak kinetic parameter (K_pat) were calculated for each patient as PET outcomes, using an automated post‐processing pipeline. Shape modeling was performed to extract the variations in bone shapes in the patients. Pearson's correlation coefficients were used to study the associations between bone shapes, PET outcomes, and patient reported pain. Direct associations between quantitative MR and PET evidence of bone remodeling were established in the acetabulum and femur. Associations of shaft thickness with SUV in the femur (p = 0.07) and K_pat in the acetabulum (p = 0.02), cam deformity with acetabular score (p = 0.09), osteophytic growth on the femur head with K_pat (p = 0.01) were observed. Pain had increased correlations with SUV in the acetabulum (p = 0.14) and femur (p = 0.09) when shaft thickness was accounted for. This study demonstrated the ability of [¹⁸F]‐NaF PET‐MRI, 3D shape modeling, and quantitative MRI to investigate cartilage‐bone interactions and bone shape features in hip OA, providing potential investigative tools to diagnose OA. © 2019 The Authors. Journal of Orthopaedic Research^® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 37:2671–2680, 2019     

Effect of cavity preparation and bone mineral density on bone‐interface densification and bone‐implant contact during press‐fit implantation of hip stems

Implant loosening and periprosthetic fracture are two major revision causes for uncemented hip stems. The chosen method of cavity preparation could play a key role for both failure mechanisms. The aim of this study was to determine the dependence of the broach type as well as patient bone mineral density (BMD) on densification and contact conditions at the bone‐implant interface. Hip stems were implanted into cadaveric femora using compaction, blunt extraction or sharp extraction broaches with computed tomography scans performed prior to broaching, after broaching and after stem implantation. Proximal periprosthetic bone densification as well as press‐fit, contact area and stem seating relative to the last broach were determined. Median bone densification was higher with the compaction and blunt extraction broaches compared to sharp extraction broaches (181% and 177%, respectively, p = 0.002). The bone densification of femora prepared with compaction broaching increased with higher BMD (R² = 0.183, p = 0.037), while stem seating decreased with higher BMD for all broach types (R² = 0.259, p = 0.001). Incomplete seated prostheses were associated with smaller press‐fit and bone‐implant contact area (R² = 0.249, p = 0.001; R² = 0.287, p < 0.001). Clinical Significance: The results suggest that compaction broaching maximizes bone densification in patients with higher bone density. However, there appears to be an increased risk of insufficient stem seating in high‐density bone that could limit the benefits for primary stability. For lower quality bone, the broach type appears to play a lesser role, but care must be taken to limit extensive stem seating which might increase periprosthetic fracture risk. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1580–1589, 2019.     

Comparison of the osteogenic capacity of minipig and human bone marrow‐derived mesenchymal stem cells

Minipigs are a recommended large animal model for preclinical testing of human orthopedic implants. Mesenchymal stem cells (MSCs) are the key repair cells in bone healing and implant osseointegration, but the osteogenic capacity of minipig MSCs is incompletely known. The aim of this study was to isolate and characterize minipig bone marrow (BM) and peripheral blood (PB) MSCs in comparison to human BM‐MSCs. BM sample was aspirated from posterior iliac crest of five male Göttingen minipigs (age 15 ± 1 months). PB sample was drawn for isolation of circulating MSCs. MSCs were selected by plastic‐adherence as originally described by Friedenstein. Cell morphology, colony formation, proliferation, surface marker expression, and differentiation were examined. Human BM‐MSCs were isolated and cultured from adult fracture patients (n = 13, age 19–60 years) using identical techniques. MSCs were found in all minipig BM samples, but no circulating MSCs could be detected. Minipig BM‐MSCs had similar morphology, proliferation, and colony formation capacities as human BM‐MSCs. Unexpectedly, minipig BM‐MSCs had a significantly lower ability than human BM‐MSCs to form differentiated and functional osteoblasts. This observation emphasizes the need for species‐specific optimization of MSC culture protocol before direct systematic comparison of MSCs between human and various preclinical large animal models can be made. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1019–1025, 2012     

Differential Effector Response of Amnion‐ and Adipose‐Derived Mesenchymal Stem Cells to Inflammation; Implications for Intradiscal Therapy

Intervertebral disc degeneration (IVDD) is a progressive condition marked by tissue destruction and inflammation. The therapeutic effector functions of mesenchymal stem cells (MSCs) makes them an attractive therapy for patients with IVDD. While several sources of MSCs exist, the optimal choice for use in the inflamed IVD remains a significant question. Adipose (AD)‐ and amnion (AM)‐derived MSCs have several advantages compared with other sources, however, no study has directly compared the impact of IVDD inflammation on their effector functions. Human MSCs were cultured in media with or without supplementation of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α at concentrations reportedly produced by IVDD cells. MSC proliferation and production of pro‐ and anti‐inflammatory cytokines were quantified following 24 and 48 h of culture. Additionally, the osteogenic and chondrogenic potential of AD‐ and AM‐MSCs was characterized via histology and biochemical analysis following 28 days of culture. In inflammatory culture, AM‐MSCs produced significantly more anti‐inflammatory IL‐10 (14.47 ± 2.39 pg/ml; p = 0.004) and larger chondrogenic pellets (5.67 ± 0.26 mm²; p = 0.04) with greater percent area staining positively for glycosaminoglycan (82.03 ± 3.26%; p < 0.001) compared with AD‐MSCs (0.00 ± 0.00 pg/ml; 2.76 ± 0.18 mm²; 34.75 ± 2.49%; respectively). Conversely, AD‐MSCs proliferated more resulting in higher cell numbers (221,000 ± 8,021 cells; p = 0.048) and produced higher concentrations of pro‐inflammatory cytokines prostaglandin E₂ (1,118.30 ± 115.56 pg/ml; p = 0.030) and IL‐1β (185.40 ± 7.63 pg/ml; p = 0.010) compared with AM‐MSCs (109,667 ± 5,696 cells; 1,291.40 ± 78.47 pg/ml; 144.10 ± 4.57 pg/ml; respectively). AD‐MSCs produced more mineralized extracellular matrix (3.34 ± 0.05 relative absorbance units [RAU]; p < 0.001) compared with AM‐MSCs (1.08 ± 0.06 RAU). Under identical inflammatory conditions, a different effector response was observed with AM‐MSCs producing more anti‐inflammatories and demonstrating enhanced chondrogenesis compared with AD‐MSCs, which produced more pro‐inflammatory cytokines and demonstrated enhanced osteogenesis. These findings may begin to help inform researchers which MSC source may be optimal for IVD regeneration. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2445–2456, 2019     

Effects of preexisting microdamage,collagen cross‐links,degree of mineralization,age, and architecture on compressive mechanical properties of elderly human vertebral trabecular bone

Previous studies have shown that the mechanical properties of trabecular bone are determined by bone volume fraction (BV/TV) and microarchitecture. The purpose of this study was to explore other possible determinants of the mechanical properties of vertebral trabecular bone, namely collagen cross‐link content, microdamage, and mineralization. Trabecular bone cores were collected from human L2 vertebrae (n = 49) from recently deceased donors 54–95 years of age (21 men and 27 women). Two trabecular cores were obtained from each vertebra, one for preexisting microdamage and mineralization measurements, and one for BV/TV and quasi‐static compression tests. Collagen cross‐link content (PYD, DPD, and PEN) was measured on surrounding trabecular bone. Advancing age was associated with impaired mechanical properties, and with increased microdamage, even after adjustment by BV/TV. BV/TV was the strongest determinant of elastic modulus and ultimate strength (r² = 0.44 and 0.55, respectively). Microdamage, mineralization parameters, and collagen cross‐link content were not associated with mechanical properties. These data indicate that the compressive strength of human vertebral trabecular bone is primarily determined by the amount of trabecular bone, and notably unaffected by normal variation in other factors, such as cross‐link profile, microdamage and mineralization. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:481–488, 2011     

Bone Fracture Toughness and Strength Correlate With Collagen Cross‐Link Maturity in a Dose‐Controlled Lathyrism Mouse Model

Collagen cross‐linking is altered in many diseases of bone, and enzymatic collagen cross‐links are important to bone quality, as evidenced by losses of strength after lysyl oxidase inhibition (lathyrism). We hypothesized that cross‐links also contribute directly to bone fracture toughness. A mouse model of lathyrism using subcutaneous injection of up to 500 mg/kg β‐aminopropionitrile (BAPN) was developed and characterized (60 animals across 4 dosage groups). Three weeks of 150 or 350 mg/kg BAPN treatment in young, growing mice significantly reduced cortical bone fracture toughness, strength, and pyridinoline cross‐link content. Ratios reflecting relative cross‐link maturity were positive regressors of fracture toughness (HP/[DHLNL + HLNL] r² = 0.208, p < 0.05; [HP + LP]/[DHNL + HLNL] r² = 0.196, p < 0.1), whereas quantities of mature pyridinoline cross‐links were significant positive regressors of tissue strength (lysyl pyridinoline r² = 0.159, p = 0.014; hydroxylysyl pyridinoline r² = 0.112, p < 0.05). Immature and pyrrole cross‐links, which were not significantly reduced by BAPN, did not correlate with mechanical properties. The effect of BAPN treatment on mechanical properties was dose specific, with the greatest impact found at the intermediate (350 mg/kg) dose. Calcein labeling was used to define locations of new bone formation, allowing for the identification of regions of normally cross‐linked (preexisting) and BAPN‐treated (newly formed, cross‐link‐deficient) bone. Raman spectroscopy revealed spatial differences attributable to relative tissue age and effects of cross‐link inhibition. Newly deposited tissues had lower mineral/matrix, carbonate/phosphate, and Amide I cross‐link (matrix maturity) ratios compared with preexisting tissues. BAPN treatment did not affect mineral measures but significantly increased the cross‐link (matrix maturity) ratio compared with newly formed control tissue. Our study reveals that spatially localized effects of short‐term BAPN cross‐link inhibition can alter the whole‐bone collagen cross‐link profile to a measureable degree, and this cross‐link profile correlates with bone fracture toughness and strength. Thus, cross‐link profile perturbations associated with bone disease may provide insight into bone mechanical quality and fracture risk. © 2014 American Society for Bone and Mineral Research.     

Combination of resveratrol‐containing collagen with adipose stem cells for craniofacial tissue‐engineering applications

Repair and regeneration of craniofacial tissues is particularly challenging because they comprise a complex structure of hard and soft tissues involved in intricate functions. This study combined collagen scaffolds and human adipose stem cells (hASCs) for oral mucosal and calvarial bone regeneration by using resveratrol (RSV), which affects the differentiation of mesenchymal stem cells. We have evaluated the effect of collagen scaffold‐containing RSV (collagen/RSV) scaffolds both in vitro and in vivo for their wound healing and bone regeneration potential. Scanning electron microscopy and immunostaining results reveal that hASCs adhere well to and proliferate on both collagen scaffolds and collagen/RSV scaffolds. Oral mucosal lesion experiments demonstrated that the collagen/RSV scaffold is more effective in wound closure and contraction than the collagen scaffold. The micro‐computed tomography (μCT) images of calvarial bone display regenerating bone in defects covered with hASCs on collagen/RSV scaffolds that are more visible than that in defects covered with hASCs on a collagen scaffolds. RSV was more effective at inducing hASC differentiation on the collagen scaffold, suggesting that collagen/RSV scaffolds can provide useful biological cues that stimulate craniofacial tissue formation.     

Defects in mesenchymal stem cell self‐renewal and cell fate determination lead to an osteopenic phenotype in Bmi‐1 null mice

In parathyroid hormone–related protein 1‐84 [PTHrP(1‐84)] knockin mice, expression of the polycomb protein Bmi‐1 is reduced and potentially can mediate the phenotypic alterations observed. We have therefore now examined the skeletal phenotype of Bmi‐1^?/? mice in vivo and also assessed the function of bone marrow mesenchymal stem cells (BM‐MSCs) from Bmi‐1^?/? mice ex vivo in culture. Neonatal Bmi‐1^?/? mice exhibited skeletal growth retardation, with reduced chondrocyte proliferation and increased apoptosis. Osteoblast numbers; gene expression of alkaline phosphatase, type I collagen, and osteocalcin; the mineral apposition rate; trabecular bone volume; and bone mineral density all were reduced significantly; however, the number of bone marrow adipocytes and Ppar‐γ expression were increased. These changes were consistent with the skeletal phenotype observed in the PTHrP(1‐84) knockin mouse. The efficiency of colony‐forming unit fibroblast (CFU‐F) formation in bone marrow cultures was decreased, and the percentage of alkaline phosphatase–positive CFU‐F and Runx2 expression were reduced. In contrast, adipocyte formation and Ppar‐γ expression in cultures were increased, and expression of the polycomb protein sirtuin (Sirt1) was reduced. Reduced proliferation and increased apoptosis of BM‐MSCs were associated with upregulation of senescence‐associated tumor‐suppressor genes, including p16, p19, and p27. Analysis of the skeletal phenotype in Bmi‐1^?/? mice suggests that Bmi‐1 functions downstream of PTHrP. Furthermore, our studies indicate that Bmi‐1 maintains self‐renewal of BM‐MSCs by inhibiting the expression of p27, p16, and p19 and alters the cell fate of BM‐MSCs by enhancing osteoblast differentiation and inhibiting adipocyte differentiation at least in part by stimulating Sirt1 expression. Bmi‐1 therefore plays a critical role in promoting osteogenesis. © 2010 American Society for Bone and Mineral Research     

Tendon‐derived progenitor cells improve healing of collagenase‐induced flexor tendinitis

Tendinitis is a common and a performance‐limiting injury in athletes. This study describes the value of intralesional tendon‐derived progenitor cell (TDPC) injections in equine flexor tendinitis. Collagenase‐induced tendinitis was created in both front superficial digital flexor (SDF) tendons. Four weeks later, the forelimb tendon lesions were treated with 1 × 10⁷ autogenous TDPCs or saline. Tendinitis was also induced by collagenase in one hind SDF tendon, to study the survival and distribution of DiI‐labeled TDPCs 1, 2, 4, and 6 weeks after injection. The remaining normal tendon was used as a “control.” Twelve weeks after forelimb TDPC injections, tendons were harvested for assessment of matrix gene expression, biochemical, biomechanical, and histological characteristics. DiI‐labeled TDPCs were abundant 1 week after injection but gradually declined over time and were undetectable after 6 weeks. Twelve weeks after TDPC injection, collagens I and III, COMP and tenomodulin mRNA levels were similar (p = 0.3) in both TDPC and saline groups and higher (p < 0.05) than normal tendon. Yield and maximal stresses of the TDPC group were significantly greater (p = 0.005) than the saline group's and similar (p = 0.6) to normal tendon. However, the elastic modulus of the TDPC and saline groups were not significantly different (p = 0.32). Histological assessment of the repair tissues with Fourier transform‐second harmonic generation imaging demonstrated that collagen alignment was significantly better (p = 0.02) in TDPC group than in the saline controls. In summary, treating collagenase‐induced flexor tendon lesions with TDPCs improved the tensile strength and collagen fiber alignment of the repair tissue. Study Design © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:2162–2171, 2016.     

Abnormal human chondrocyte morphology is related to increased levels of cell‐associated IL‐1β and disruption to pericellular collagen type VI

Early osteoarthritis (OA) is poorly understood, but abnormal chondrocyte morphology might be important. We studied IL‐1β and pericellular collagen type VI in morphologically normal and abnormal chondrocytes. In situ chondrocytes within explants from nondegenerate (grade 0/1) areas of human tibial plateaus (n = 21) were fluorescently labeled and visualized [2‐photon laser scanning microscopy (2PLSM)]. Normal chondrocytes exhibited a “smooth” membrane surface, whereas abnormal cells were defined as demonstrating ≥1 cytoplasmic process. Abnormal chondrocytes were further classified by number and average length of cytoplasmic processes/cell. IL‐1β or collagen type VI associated with single chondrocytes were visualized by fluorescence immuno‐histochemistry and confocal laser scanning microscopy (CLSM). Fluorescence was quantified as the number of positive voxels (i.e., 3D pixels with fluorescence above baseline)/cell. IL‐1β‐associated fluorescence increased between normal and all abnormal cells in the superficial (99.7 ± 29.8 [11 (72)] vs. 784 ± 382 [15 (132)]; p = 0.04, positive voxels/cell) and deep zones (66.5 ± 29.4 [9 (64)] vs. 795 ± 224 [9 (56)]; p = 0.006). There was a correlation (r² = 0.988) between the number of processes/cell (0–5) and IL‐1β, and an increase particularly with short processes (≤5 µm; p = 0.022). Collagen type VI coverage and thickness decreased (p < 0.001 and p = 0.005, respectively) with development of processes. Abnormal chondrocytes in macroscopically nondegenerate cartilage demonstrated a marked increase in IL‐1β and loss of pericellular type VI collagen, changes that could lead to cartilage degeneration. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1507–1514, 2010     

Increase in cell migration and angiogenesis in a composite silk scaffold for tissue‐engineered ligaments

The purpose of this study was to evaluate the biocompatibility of silk and collagen‐hyaluronan (HA) in vitro by assessing anterior cruciate ligament (ACL) cell and T‐lymphocyte cultures on scaffolds. The use of composite scaffolds as artificial ligaments in ACL reconstruction and their effects on angiogenesis were evaluated in vivo. The silk scaffold was knitted by hand and dry coated with collagen‐HA, whereas the composite silk scaffold was made by covering a silk scaffold with a lyophilized collagen‐HA substrate. The initial attachment and proliferation of human ACL cells on the composite silk scaffold was superior to the attachment and proliferation observed on the silk scaffold. The immune response was higher in both scaffolds after 72 h (p < 0.05) compared with the control culture condition without scaffolding, as assessed by T‐lymphocyte cultures in vitro. There was no significant difference in the immune response in vitro between the silk and composite silk scaffolds. Silk and composite silk scaffolds were implanted as artificial ligaments in ACLs removed from the knees of dogs, and they were harvested 6 weeks after implantation. On gross examination, the onset of an inflammatory tissue reaction, such as synovitis, was seen in both the silk scaffold and the composite silk scaffold groups. An histological evaluation of the artificial ligament implants revealed the presence of monocytes in the silk composite scaffold and the absence of giant cells in all cases. MT staining in the composite silk scaffold‐grafted group showed granulation tissue consisting of fibroblasts, lymphocytes, monocytes, and collagen fibers. In addition, CD31 staining revealed the formation of new blood vessels. On the other hand, no reparative tissues, such as blood vessels, collagen, and cells, were observed in the silk scaffold‐grafted group. These results suggest that the lyophilized collagen‐HA substrate is biocompatible in vitro and enhances new blood vessel and cell migration in vivo. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 495–503, 2009     

Erythrocytes inhibit ligament fibroblast proliferation in a collagen scaffold

In this work, we hypothesized that the concentration of erythrocytes in a provisional scaffold would have a significant effect on three of the major biological processes occurring in early wound healing. ACL fibroblast proliferation, collagen production, and scaffold contraction were measured in collagen gels containing fibroblasts and erythrocytes in subphysiologic (1 × 10⁸ erythrocytes/ml), physiologic (1 × 10⁹ erythrocytes/ml), and supraphysiologic (1 × 10¹⁰ erythrocytes/ml) concentrations. Fibroblast‐seeded gels containing only platelet‐poor plasma were used as a control group. All gels were cultured for 1, 14, and 21 days. DNA, ELISA for procollagen and scaffold size measurements were used to quantify the three above parameters of wound healing. Samples with concentrations of erythryocytes lower than that in whole blood stimulated greater fibroblast proliferation and scaffold contraction than those with erythrocyte concentrations similar to that in whole blood (p < 0.027; p < 0.03). Increasing the erythrocyte concentration over that in the whole blood stimulated fibroblast collagen production (p < 0.009) and limited scaffold contraction (p < 0.031). Further work examining the role of the erythrocyte in the early provisional scaffold is warranted. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1361–1366, 2011     

Evaluation of a starch‐based double layer scaffold for bone regeneration in a rat model

Damages in the maxillofacial bones are frequent in humans following trauma, metabolic diseases, neoplasia, or inflammatory processes. Many of the available treatments to regenerate bone are often ineffective. The goal of this work was to assess the in vivo behavior of an innovative double‐layered scaffold based on a blend of starch and polycaprolactone (SPCL) that comprises a membrane obtained by solvent casting, which aims to act as a guided tissue regeneration membrane, and a wet‐spun fiber mesh (in some cases functionalized with osteoconductive silanol groups) targeting bone regeneration. The behavior of the double layer scaffold, functionalized with silanol groups (SPCL‐Si) or without (SPCL), was assessed in a mandibular rodent model and compared to a commercial collagen membrane (positive control) and to empty defects (negative control). After 8 weeks of implantation, the micro‐computed tomography and the histomorphometric analysis revealed that the SPCL‐Si scaffolds induced significantly higher new bone formation compared to the collagen membrane and to the empty defects, although they had a similar performance when compared to the SPCL scaffolds. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:904–909, 2014.     

Effects of platelet‐rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: A placebo‐controlled experimental study

Tendon injuries are notorious for their slow and functionally inferior healing. Intratendinous application of platelet‐rich plasma (PRP) has been reported to stimulate the repair process of tendon injuries, but there is little conclusive evidence for its effectiveness. A placebo‐controlled experimental trial was performed to test the hypothesis that a single intratendinous PRP treatment enhances the quality of tendon repair, as evidenced by improved biochemical, biomechanical, and histological tissue properties. In six horses, tendon lesions were created surgically in the Superficial Digital Flexor Tendons (SDFT) of both front limbs, one of which was treated with PRP and the other with saline. After 24 weeks, the tendons were harvested for biochemical, biomechanical, and histological evaluations. Collagen, glycosaminoglycan, and DNA content (cellularity) was higher in PRP‐treated tendons (p = 0.039, 0.038, and 0.034, respectively). The repair tissue in the PRP group showed a higher strength at failure (p = 0.021) and Elastic Modulus (p = 0.019). Histologically, PRP‐treated tendons featured better organization of the collagen network (p = 0.031) and signs of increased metabolic activity (p = 0.031). It was concluded that PRP increases metabolic activity and seems to advance maturation of repair tissue over nontreated experimentally induced tendon lesions, which suggests that PRP might be beneficial in the treatment of clinical tendon injuries. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:211–217, 2010