Low‐intensity pulsed ultrasound accelerates fracture healing by stimulation of recruitment of both local and circulating osteogenic progenitors

We investigated the effect of low‐intensity pulsed ultrasound (LIPUS) on the homing of circulating osteogenic progenitors to the fracture site. Parabiotic animals were formed by surgically conjoining a green fluorescent protein (GFP) mouse and a syngeneic wild‐type mouse. A transverse femoral fracture was made in the contralateral hind limb of the wild‐type partner. The fracture site was exposed to daily LIPUS in the treatment group. Animals without LIPUS treatment served as the control group. Radiological assessment showed that the hard callus area was significantly greater in the LIPUS group than in the control group at 2 and 4 weeks post‐fracture. Histomorphometric analysis at the fracture site showed a significant increase of GFP cells in the LIPUS group after 2 weeks (7.5%), compared to the control group (2.4%) (p < 0.05). The LIPUS group exhibited a significantly higher percentage of GFP cells expressing alkaline phosphatase (GFP/AP) than the control group at 2 weeks post‐fracture (5.9%, 0.3%, respectively, p < 0.05). There was no significant difference in the percentage of GFP/AP cells between the LIPUS group (2.0%) and the control group (1.4%) at 4 weeks post‐fracture. Stromal cell derived factor‐1 and CXCR4 were immunohistochemically identified at the fracture site in the LIPUS group. These data indicate that LIPUS induced the homing of circulating osteogenic progenitors to the fracture site for possible contribution to new bone formation. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1516–1521, 2012     

Low‐magnitude high‐frequency vibration accelerates callus formation,mineralization, and fracture healing in rats

Fracture healing is a biological regenerative process that follows a well‐orchestrated sequence. Most healing is uneventful and enhancement of normal fracture healing is not commonly done, although it is clinically important in the recovery and regain of functions after fracture. This study investigated the osteogenic effect of low‐magnitude high‐frequency vibration (LMHFV, 35 Hz, 0.3 g) on the enhancement of fracture healing in rats with closed femoral shaft fracture by comparing with sham‐treated control. Assessments with plain radiography, micro‐CT as well as histomorphometry showed that the amount of callus was significantly larger (p = 0.001 for callus area, 2 weeks posttreatment); the remodeling of the callus into mature bone was significantly faster (p = 0.039, 4 weeks posttreatment) in the treatment group. The mechanical strength of the healed fracture in the treatment group at 4 weeks was significantly greater (p < 0.001). The results showed the acceleration of callus formation, mineralization, and fracture healing in the treatment group. It is concluded that LMHFV enhances healing in the closed femoral shaft fracture in rats. The potential clinical advantages shall be confirmed in the subsequent clinical trials. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 458–465, 2009     

SDF‐1/CXCR4 Axis in Tie2‐Lineage Cells Including Endothelial Progenitor Cells Contributes to Bone Fracture Healing

CXC chemokine receptor 4 (CXCR4) is a specific receptor for stromal‐derived‐factor 1 (SDF‐1). SDF‐1/CXCR4 interaction is reported to play an important role in vascular development. On the other hand, the therapeutic potential of endothelial progenitor cells (EPCs) in fracture healing has been demonstrated with mechanistic insight of vasculogenesis/angiogenesis and osteogenesis enhancement at sites of fracture. The purpose of this study was to investigate the influence of the SDF‐1/CXCR4 pathway in Tie2‐lineage cells (including EPCs) in bone formation. We created CXCR4 gene conditional knockout mice using the Cre/loxP system and set two groups of mice: Tie2‐Cre^ER CXCR4 knockout mice (CXCR4^?/?) and wild‐type mice (WT). We report here that in vitro, EPCs derived from of CXCR4^?/? mouse bone marrow demonstrated severe reduction of migration activity and EPC colony‐forming activity when compared with those derived from WT mouse bone marrow. In vivo, radiological and morphological examinations showed fracture healing delayed in the CXCR4^?/? group and the relative callus area at weeks 2 and 3 was significantly smaller in CXCR4^?/? group mice. Quantitative analysis of capillary density at perifracture sites also showed a significant decrease in the CXCR4^?/? group. Especially, CXCR4^?/?group mice demonstrated significant early reduction of blood flow recovery at fracture sites compared with the WT group in laser Doppler perfusion imaging analysis. Real‐time RT‐PCR analysis showed that the gene expressions of angiogenic markers (CD31, VE‐cadherin, vascular endothelial growth factor [VEGF]) and osteogenic markers (osteocalcin, collagen 1A1, bone morphogenetic protein 2 [BMP2]) were lower in the CXCR4^?/? group. In the gain‐of‐function study, the fracture in the SDF‐1 intraperitoneally injected WT group healed significantly faster with enough callus formation compared with the SDF‐1 injected CXCR4^?/? group. We demonstrated that an EPC SDF‐1/CXCR4 axis plays an important role in bone fracture healing using Tie2‐Cre^ER CXCR4 conditional knockout mice. © 2014 American Society for Bone and Mineral Research.     

Collagen‐containing scaffolds enhance attachment and proliferation of non‐cultured bone marrow multipotential stromal cells

Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen‐containing bovine bone scaffold (Orthoss^® Collagen) with a non‐collagen‐containing bovine bone scaffold, Orthoss^®. Another collagen‐containing synthetic scaffold, Vitoss^® was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit‐fibroblast assay and flow‐cytometry. The number of BM MSCs initially attached to Orthoss^® Collagen and Vitoss^® was similar but greater than Orthoss^® (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss^® Collagen and Vitoss^® after 2‐week culture was also higher compared to Orthoss^® (p = 0.010 and p = 0.023, respectively). Interestingly, collagen‐containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture‐expanded MSCs on Orthoss^® collagen and Vitoss^® was greater compared to Orthoss^® (p = 0.047 and p = 0.004, respectively). Collectively, collagen‐containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:597–606, 2016.     

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     

Low‐magnitude high‐frequency vibration enhances gene expression related to callus formation,mineralization and remodeling during osteoporotic fracture healing in rats

Low magnitude high frequency vibration (LMHFV) has been shown to improve anabolic and osteogenic responses in osteoporotic intact bones and during osteoporotic fracture healing; however, the molecular response of LMHFV during osteoporotic fracture healing has not been investigated. It was hypothesized that LMHFV could enhance osteoporotic fracture healing by regulating the expression of genes related to chondrogenesis (Col‐2), osteogenesis (Col‐1) and remodeling (receptor activator for nuclear factor‐ κ B ligand (RANKL) and osteoproteger (OPG)). In this study, the effects of LMHFV on both osteoporotic and normal bone fracture healing were assessed by endpoint gene expressions, weekly radiographs, and histomorphometry at weeks 2, 4 and 8 post‐treatment. LMHFV enhanced osteoporotic fracture healing by up‐regulating the expression of chondrogenesis‐, osteogenesis‐ and remodeling‐related genes (Col‐2 at week 4 (p = 0.008), Col‐1 at week 2 and 8 (p < 0.001and p = 0.008) and RANKL/OPG at week 8 (p = 0.045)). Osteoporotic bone had a higher response to LMHFV than normal bone and showed significantly better results as reflected by increased expression of Col‐2 and Col‐1 at week 2 (p < 0.001 for all), larger callus width at week 2 (p = 0.001), callus area at week 1 and 5(p < 0.05 for all) and greater relative area of osseous tissue (p = 0.002) at week 8. This study helps to understand how LMHFV regulates gene expression of callus formation, mineralization and remodeling during osteoporotic fracture healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1572–1579, 2014.     

Effects of blockade of endogenous Gi signaling in Tie2‐expressing cells on bone formation in a mouse model of heterotopic ossification

Available evidence indicates that some Tie2‐expressing (Tie2⁺) cells serve as multipotent progenitors that have robust BMP‐dependent osteogenic activity and mediate heterotopic ossification (HO). Since signaling through the G protein G_i is required for cell motility, we hypothesized that blockade of endogenous G_i signaling in Tie2⁺ cell populations would prevent HO formation. Blockade of G_i signaling in Tie2⁺ cells was accomplished in transgenic mice with expression of pertussis toxin (PTX) under the control of the Tie2 promoter (Tie2⁺/PTX⁺). Bone formation within HOs was evaluated 2 weeks after BMP injection. Expression of PTX in Tie2⁺ cells significantly reduced the bone volume (BV) of HOs in male and female mice. Orthotopic bones were assessed at the distal femur and expression of PTX significantly increased trabecular bone fractional volume and bone formation rate in females only. In adult Tie2⁺/GFP⁺ mice, GFP⁺ cells appeared both inside and at the surfaces of bone tissue within HOs and in orthotopic bones. In summary, blockade of G_i signaling in Tie2⁺ cells reduced the accrual of HOs and stimulated osteogenesis in orthotopic bones. Targeting of G_i protein coupled receptors in Tie2⁺ cells may be a novel therapeutic strategy in states of abnormal bone formation such as osteoporosis and HO. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1212–1217, 2015.     

CCL2 But Not CCR2 Is Required for Spontaneous Articular Cartilage Regeneration Post‐Injury

The role of the inflammatory response in articular cartilage degeneration and/or repair is often debated. Chemokine networks play a critical role in directing the recruitment of immune cells to sites of injury and have been shown to regulate cell behavior. In this study, we investigated the role of the CCL2/CCR2 signaling axis in cartilage regeneration and degeneration. CCL2^?/?, CCR2^?/?, CCL2^?/?CCR2^?/?, and control (C57) mice were subjected to full‐thickness cartilage defect (FTCD) injuries (n = 9/group) within the femoral groove. Cartilage regeneration at 4 and 12 weeks post‐FTCD was assessed using a 14‐point histological scoring scale. Mesenchymal stem cells (MSCs) (Sca‐1⁺, CD140a⁺), macrophages (M1:CD38⁺, M2:CD206⁺, and M0:F4/80⁺) and proliferating cells (Ki67⁺) were quantified within joints using immunofluorescence. The multi‐lineage differentiation capacity of Sca1⁺ MSCs was determined for all mouse strains. ACL transection (ACL‐x) was employed to determine if CCL2^?/?CCR2^?/? mice were protected against osteoarthritis (OA) (n = 6/group). Absence of CCR2, but not CCL2 nor both (CCL2 and CCR2), enhanced spontaneous articular cartilage regeneration by 4 weeks post‐FTCD. Furthermore, increased chondrogenesis was observed in MSCs derived from CCR2^?/? mice. CCL2 deficiency promoted MSC homing to the adjacent synovium and FTCD at both 4 and 12 weeks post‐injury; with no MSCs present at the surface of the FTCD in the remaining strains. Lower OA scores were observed in CCL2^?/?CCR2^?/? mice at 12 weeks post‐ACL‐x compared with C57 mice. Our findings demonstrate an inhibitory role for CCR2 in cartilage regeneration after injury, while CCL2 is required for regeneration, acting through a CCR2 independent mechanism. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2561–2574, 2019     

Effects of Interleukin‐6 Ablation on Fracture Healing in Mice

This study examined the impact of an interleukin‐6 (IL‐6) knockout on fracture healing in terms of histological and biomechanical responses. Following IACUC approval, tibial fractures were produced in 4‐ to 6‐week‐old IL‐6 knockouts (n = 35) and wild‐type mice (n = 36) and harvested along with contralateral limbs at 2 and 6 weeks postsurgery. Histology quantified stage of healing, lymphocyte infiltration, TRAP+ cells, and osteocalcin deposition. Bend testing established maximum load and stiffness. Based on normality assessments, Mann–Whitney U or independent t‐tests were used for data analysis using a p‐value threshold of 0.05. Stage of healing, lymphocyte infiltration, and osteocalcin deposition were similar for all time points (p ≥ 0.243). TRAP+ cell counts were reduced approximately 10‐fold in the knockout at 2 weeks (p = 0.015) but were similar at 6 weeks (p = 0.689). Force‐to‐failure in knockouts was approximately 40% that of wild‐type mice at 2 weeks (p = 0.040) but similar at 6 weeks (p = 0.735). Knockout bone was about 25% less stiff at 2 weeks but approximately 60% stiffer at 6 weeks (p ≥ 0.110). The absence of IL‐6 during early fracture healing significantly reduced osteoclastogenesis and impaired callus strength. By 6 weeks, most histological and biomechanical parameters were similar to fractures in wild‐type bone. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1437–1442, 2011     

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.     

Long‐term administration of AMD3100, an antagonist of SDF‐1/CXCR4 signaling,alters fracture repair

Fracture healing involves rapid stem and progenitor cell migration, homing, and differentiation. SDF‐1 (CXCL12) is considered a master regulator of CXCR4‐positive stem and progenitor cell trafficking to sites of ischemic (hypoxic) injury and regulates their subsequent differentiation into mature reparative cells. In this study, we investigated the role of SDF‐1/CXCR4 signaling in fracture healing where vascular disruption results in hypoxia and SDF‐1 expression. Mice were injected with AMD3100, a CXCR4 antagonist, or vehicle twice daily until euthanasia with the intent to impair stem cell homing to the fracture site and/or their differentiation. Fracture healing was evaluated using micro‐computed tomography, histology, quantitative PCR, and mechanical testing. AMD3100 administration resulted in a significantly reduced hyaline cartilage volume (day 14), callus volume (day 42) and mineralized bone volume (day 42) and reduced expression of genes associated with endochondral ossification including collagen Type 1 alpha 1, collagen Type 2 alpha 1, vascular endothelial growth factor, Annexin A5, nitric oxide synthase 2, and mechanistic target of rapamycin. Our data suggest that the SDF‐1/CXCR4 signaling plays a central role in bone healing possibly by regulating the recruitment and/or differentiation of stem and progenitor cells. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1853–1859, 2012     

Cell line IDG‐SW3 replicates osteoblast‐to‐late‐osteocyte differentiation in vitro and accelerates bone formation in vivo

Osteocytes are the most abundant cells in bone yet are the most challenging to study because they are embedded in a mineralized matrix. We generated a clonal cell line called IDG‐SW3 (for Immortomouse/Dmp1‐GFP‐SW3) from long‐bone chips from mice carrying a Dmp1 promoter driving GFP crossed with the Immortomouse, which expresses a thermolabile SV40 large T antigen regulated by interferon γ (IFN‐γ). Cells from these mice can be expanded at 33 °C in the presence of IFN‐γ and then allowed to resume their original phenotype at 37 °C in the absence of IFN‐γ. IDG‐SW3 cells are Dmp1‐GFP^? and T antigen⁺ under immortalizing conditions but Dmp1‐GFP⁺ and T antigen^? under osteogenic conditions. Like osteoblasts, they express alkaline phosphatase and produce and mineralize a type 1 collagen matrix containing calcospherulites. Like early osteocytes, they express E11/gp38, Dmp1, MEPE, and Phex. Like late osteocytes, they develop a dendritic morphology and express SOST/sclerostin and fibroblast growth factor 23 (FGF‐23), regulated by parathyroid hormone (PTH) and 1,25‐dihydroxyvitamin D₃. When cultured on 3D matrices, they express Dmp1‐GFP and sclerostin. When the 3D cultures are implanted in calvarial defects in vivo, they accelerate bone healing. This cell line should prove useful for studying osteoblast‐to‐osteocyte transition, mechanisms for biomineralization, osteocyte function, and regulation of SOST/sclerostin and FGF‐23. © 2011 American Society for Bone and Mineral Research     

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.

     

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     

Assessment of contrast‐enhanced computed tomography for imaging of cartilage during fracture healing

Assessment of the early stages of fracture healing via X‐rays and computed tomography is limited by the low radio‐opacity of cartilage. We validated a method of contrast‐enhanced computed tomography (CECT) for non‐destructive identification of cartilage within a healing fracture callus. Closed, stabilized fractures in femora of C57BL/6 mice were harvested on post‐operative day 9.5 and imaged ex vivo with micro‐computed tomography (µCT) before and after incubation in a cationic contrast agent that preferentially accumulates in cartilage due to the high concentration of sulfated glycosaminoglycans in the tissue. Co‐registration of the pre‐ and post‐incubation images, followed by image subtraction, enabled two‐ and three‐dimensional delineation of mineralized tissue, soft callus, and cartilage. The areas of cartilage and callus identified with CECT were compared to those identified with the gold‐standard method of histomorphometry. No difference was found between the areas of cartilage measured by the two methods (p = 0.999). Callus area measured by CECT was smaller than, but strongly predictive of (R² = 0.80, p < 0.001), the corresponding histomorphometric measurements. CECT also enabled qualitative identification of mineralized cartilage. These findings indicate that the CECT method provides accurate, quantitative, and non‐destructive visualization of the shape and composition of the fracture callus, even during the early stages of repair when little mineralized tissue is present. The non‐destructive nature of this method would allow subsequent analyses, such as mechanical testing, to be performed on the callus, thus enabling higher‐throughput, comprehensive investigations of bone healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 567–573, 2013     

CCL2/MCP‐1 and CXCL12/SDF‐1 blockade by L‐aptamers improve pancreatic islet engraftment and survival in mouse

The blockade of pro‐inflammatory mediators is a successful approach to improve the engraftment after islet transplantation. L‐aptamers are chemically synthesized, nonimmunogenic bio‐stable oligonucleotides that bind and inhibit target molecules conceptually similar to antibodies. We aimed to evaluate if blockade‐aptamer‐based inhibitors of C‐C Motif Chemokine Ligand 2/monocyte chemoattractant protein‐1 (CCL2/MCP‐1) and C‐X‐C Motif Chemokine Ligand 12/stromal cell‐derived factor‐1 (CXCL12/SDF‐1) are able to favor islet survival in mouse models for islet transplantation and for type 1 diabetes. We evaluated the efficacy of the CCL2‐specific mNOX‐E36 and the CXCL12‐specific NOX‐A12 on islet survival in a syngeneic mouse model of intraportal islet transplantation and in a multiple low doses of streptozotocin (MLD‐STZ) diabetes induction model. Moreover, we characterized intrahepatic infiltrated leukocytes by flow cytometry before and 3 days after islet infusion in presence or absence of these inhibitors. The administration for 14 days of mNOX‐E36 and NOX‐A12 significantly improved islet engraftment, either compound alone or in combination. Intrahepatic islet transplantation recruited CD45⁺ leucocytes and more specifically CD45+/CD11b+ mono/macrophages; mNOX‐E36 and NOX‐A12 treatments significantly decreased the recruitment of inflammatory monocytes, CD11b⁺/Ly6C^high/CCR2⁺ and CD11b⁺/Ly6C^high/CXCR4⁺ cells, respectively. Additionally, both L‐aptamers significantly attenuated diabetes progression in the MLD‐STZ model. In conclusion, CCL2/MCP‐1 and CXCL12/SDF‐1 blockade by L‐aptamers is an efficient strategy to improve islet engraftment and survival.     

Impact of Basiliximab on regulatory T‐cells early after kidney transplantation: down‐regulation of CD25 by receptor modulation

Monoclonal anti‐CD25‐antibodies are successfully applied in organ transplantation to reduce the incidence of acute graft rejection. However, targeting the CD25 molecule might not only affect activated T‐cells but also regulatory T‐cells (T_regs) constitutively expressing the CD4⁺CD25⁺CD127^lowFoxP3⁺ phenotype. In this study, we investigated the influence of the anti‐CD25‐antibody Basiliximab on the frequency of T_regs early after kidney transplantation comparing individuals receiving/not receiving induction therapy (n = 14 and n = 7). Following Basiliximab administration, a distinct loss of CD4⁺CD25^high T‐cells was observed lasting for at least 6 weeks. This was not accompanied by a disappearance of the entire CD4⁺CD25⁺FoxP3⁺ T_regs but rather a decreased expression density of CD25 on the latter. In addition, a transient rise in CD4⁺CD25^?FoxP3⁺ T‐cells was found which expressed the CD127^low phenotype. Thus, a phenotypic shift of T_regs from the CD25⁺ to the CD25^? compartment was suggested. This was supported by in vitro findings showing that the disappearance of CD4⁺CD25^high cells in the presence of Basiliximab was due to down‐regulation of CD25 expression meanwhile the suppressive function of these cells was maintained. In conclusion, Basiliximab therapy directly affects CD4⁺CD25⁺CD127^lowFoxP3⁺ T_regs but does not seem to be associated with functional consequences. Thus, it is unlikely that Basiliximab treatment negatively influences strategies involving T_regs to promote tolerance after organ transplantation.     

Investigating the role of hematopoietic stem and progenitor cells in regulating the osteogenic differentiation of mesenchymal stem cells in vitro

Significant progress has been made in understanding the hematopoietic supportive capacity of both mesenchymal stem cells (MSCs) and osteogenic cells in maintaining hematopoietic stem and progenitor cells (HSPCs) in vitro. However the role of HSPCs in regulating their bone marrow niche environment through influencing the function of neighboring cell populations to complete this reciprocal relationship is not well understood. In this study, we investigated the influence of HSPCs on the osteogenic differentiation of MSCs in vitro, using a highly enriched population of hematopoietic cells with the phenotype c‐Kit⁺Sca‐1⁺Lineage^? (KSL) and bone marrow derived mesenchymal stromal cells in direct contact co‐culture in medium with or without the addition of the osteogenic supplement dexamethasone. The data suggest that a low dose of HSPCs in co‐culture with MSCs in combination with dexamethasone treatment accelerates the osteogenic progression of MSCs, as evidenced in the earlier peak in alkaline phosphatase activity and enhanced calcium deposition compared to cultures of MSCs alone. We observed a longer persistence of functional primitive hematopoietic stem and progenitor cells in the population treated with dexamethasone, and this observation was positively correlated with enhanced osteogenic differentiation of MSCs. Therefore, our findings further support the concept that HSPCs are actively involved in regulating the development and maintenance of the stem cell niche environment in which they reside. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1544–1553, 2011     

Mesenchymal stem cells and insulin‐like growth factor‐I gene‐enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons

Tendinitis remains a catastrophic injury among athletes. Mesenchymal stem cells (MSCs) have recently been investigated for use in the treatment of tendinitis. Previous work has demonstrated the value of insulin‐like growth factor‐I (IGF‐I) to stimulate cellular proliferation and tendon fiber deposition in the core lesion of tendinitis. This study examined the effects of MSCs, as well as IGF‐I gene‐enhanced MSCs (AdIGF‐MSCs) on tendon healing in vivo. Collagenase‐induced bilateral tendinitis lesions were created in equine flexor digitorum superficialis tendons (SDFT). Tendons were treated with 10 × 10⁶ MSCs or 10 × 10⁶ AdIGF‐MSCs. Control limbs were injected with 1 mL of phosphate‐buffered saline (PBS). Ultrasound examinations were performed at t = 0, 2, 4, 6, and 8 weeks. Horses were euthanized at 8 weeks and SDFTs were mechanically tested to failure and evaluated for biochemical composition and histologic characteristics. Expression of collagen types I and III, IGF‐I, cartilage oligomeric matrix protein (COMP), matrix metalloproteinase‐3 (MMP‐3), matrix metalloproteinase‐13 (MMP‐13), and aggrecanase‐1 (ADAMTS‐4) were similar in MSC and control tendons. Both MSC and AdIGF‐MSC injection resulted in significantly improved tendon histological scores. These findings indicate a benefit to the use of MSCs and AdIGF‐MSCs for the treatment of tendinitis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1392–1398, 2009     

Thrombospondin‐2 spatiotemporal expression in skeletal fractures

Fracture healing is a complex process that relies heavily on the carefully orchestrated expansion and differentiation of periosteal mesenchymal progenitor cells (MSC). Identification of new markers for periosteal MSCs is essential for the development of fracture therapeutics. Expression of the matricellular protein thrombospondin‐2 (TSP2) increases during early fracture healing; however, it is currently unknown what cell population expresses TSP2. Using a TSP2 GFP reporter mouse and a stabilized murine fracture model, we characterized the expression of TSP2 during the inflammatory, soft callus formation, and hard callus formation phases of fracture healing. In addition, using TSP2 GFP positive cells harvested from reporter mouse cells, we characterized the cell population using flow cytometry and colony formation assays. In uninjured diaphyseal bone, we observed TSP2 expression in the cells located along the inner periosteum. We also observed a population of TSP2 expressing cells in undifferentiated regions of early fracture callus and along the periphery of the callus. Later in callus development, TSP2 cells were broadly distributed in the undifferentiated callus, but GFP was not expressed by chondrocytes. Flow cytometry confirmed that the majority of TSP2 expressing cells were positive for traditional murine MSC markers. Our in vitro assays further supported these findings by demonstrating all adherent and colony‐forming cells expressed TSP2. Taken together, our results suggest that TSP2 is expressed by undifferentiated MSCs, but downregulated in chondrocytes. Clinical significance: expression of the matricellular protein TSP2 is a promising new marker to identify MSCs in early fracture healing.