CD45-antibody-drug conjugate clears tissue resident myeloid cells from their niches enabling therapeutic adoptive cell transfer.

Tissue resident myeloid cells (TRM) in adults have highly variable lifespans and may be derived from early embryonic yolk sac, fetal liver or bone marrow. Some of these TRM are known pathogenic participants in congenital and acquired diseases. Myeloablative conditioning and hematopoietic stem cell transplant can replace long-lived brain TRM resulting in clinical improvements in metabolic storage diseases. With the advent of antibody-drug-conjugate (ADC) targeted cell killing as a cell selective means of transplant conditioning, we assessed the impact of anti-CD45-ADC on TRM in multiple tissues. Replacement of TRM ranged from 40 to 95 percent efficiencies in liver, lung, and skin tissues, after a single anti-CD45-ADC dose and bone marrow hematopoietic cell transfer. Of note, the population size of TRM in tissues returned to pre-treatment levels suggesting a regulated control of TRM abundance. As expected, brain, microglia were not affected, but brain monocytes and macrophages were 50% replaced. Anti-CD45-ADC and adoptive cell transfer were then tested in the chronic acquired condition, atherosclerosis exacerbated by Tet2 mutant clonal hematopoiesis. Plaque resident myeloid cells were efficiently replaced with anti-CD45-ADC and wild-type bone marrow cells. Notably, this reduced existent atherosclerotic plaque burden. Overall, these results indicate that anti-CD45-ADC clears both HSC and TRM niches enabling cell replacement to achieve disease modification in a resident myeloid cell driven disease.

Clearing and replacing tissue-resident myeloid cells with an anti-CD45 antibody-drug conjugate.

Tissue-resident myeloid (TRM) cells in adults have highly variable lifespans, and may be derived from early embryonic yolk sac, fetal liver, or bone marrow. Some of these TRM cells are known pathogenic participants in congenital and acquired diseases. Myeloablative conditioning and hematopoietic stem cell transplantation can replace long-lived brain TRM cells, resulting in clinical improvements in metabolic storage diseases. With the advent of antibody-drug conjugate (ADC)-targeted cell killing as a cell-selective means of transplant conditioning, we assessed the impact of anti-CD45-ADC on TRM cells in multiple tissues. Replacement of TRM cells ranged from 40% to 95% efficiencies in liver, lung, and skin tissues, after a single anti-CD45-ADC dose and bone marrow hematopoietic cell transfer. Of note, the population size of TRM cells in tissues returned to pretreatment levels, suggesting a regulated control of TRM cell abundance. As expected, brain microglia were not affected, but brain monocytes and macrophages were 50% replaced. Anti-CD45-ADC and adoptive cell transfer were then tested in the chronic acquired condition, atherosclerosis exacerbated by Tet2 mutant clonal hematopoiesis. Plaque-resident myeloid cells were efficiently replaced with anti-CD45-ADC and wild-type bone marrow cells. Notably, this reduced existent atherosclerotic plaque burden. Overall, these results indicate that the anti-CD45-ADC clears both hematopoietic stem and TRM cells from their niches, enabling cell replacement to achieve disease modification in a resident myeloid cell-driven disease.

VEGF-A regulates angiogenesis during osseointegration of Ti implants via paracrine/autocrine regulation of osteoblast response to hierarchical microstructure of the surface.

Establishment of a patent vasculature at the bone-implant interface plays a significant role in determining overall success of orthopedic and dental implants. Osteoblasts produce vascular endothelial growth factor-A (VEGF-A), an important regulator of angiogenesis during bone formation and healing, and the amount secreted is sensitive to titanium (Ti) surface microtopography and surface energy. The purpose of this study was to determine if surface properties modulate cellular response to VEGF-A. MG63 osteoblast-like cells were transfected with shRNA targeting VEGF-A at >80% knockdown. Cells stably silenced for VEGF-A secreted reduced levels of osteocalcin, osteoprotegerin, FGF-2, and angiopoietin-1 when cultured on grit-blasted/acid-etched (SLA) and hydrophilic SLA (modSLA) Ti surfaces and conditioned media from these cultures caused reduced angiogenesis in an endothelial tubule formation assay. Treatment of MG63 cells with 20 ng/mL rhVEGF-A165 rescued production in silenced cells and increased production of osteocalcin, osteoprotegerin, FGF-2, and angiopoietin-1, with greatest effects on control cells cultured on modSLA. Addition of a neutralization antibody against VEGF receptor 2 (VEGFR2; Flk-1) resulted in a significant increase in VEGF-A production. Overall, this study indicates that VEGF-A has two roles in osseointegration: enhanced angiogenesis and an autocrine/paracrine role in maturation of osteoblast-like cells in response to Ti surface properties. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 423-433, 2019.

Effects of Tunable Keratin Hydrogel Erosion on Recombinant Human Bone Morphogenetic Protein 2 Release, Bioactivity, and Bone Induction.

IMPACT STATEMENT: Recombinant human bone morphogenetic protein 2 (rhBMP-2) delivery from collagen sponges for bone formation is an important clinical example of growth factors in tissue engineering. Side effects from rhBMP-2 burst release and rapid collagen resorption have led to investigation of alternative carriers. Here, keratin carriers with tunable erosion rates were formulated by varying disulfide crosslinking via ratios of oxidatively (keratose) to reductively (kerateine) extracted keratin. In vitro rhBMP-2 bioactivity increased with kerateine content, reaching levels greater than with collagen. Heterotopic bone formation in a mouse model depended on the keratin formulation, highlighting the importance of the growth factor carrier.

Role of Wnt11 during Osteogenic Differentiation of Human Mesenchymal Stem Cells on Microstructured Titanium Surfaces.

Successful osseointegration of an endosseous implant involves migration and differentiation of mesenchymal stem cells (MSCs) on the implant surface. Micro-structured, hydrophilic titanium surfaces direct MSCs to undergo osteoblastic differentiation in vitro, in the absence of media additives commonly used in cultures grown on tissue culture polystyrene (TCPS). This process involves non-canonical Wnt5a, in contrast to canonical Wnt3a typically credited with osteoblastic differentiation on TCPS. Wnt proteins have been implicated in morphological development and tissue patterning, suggesting that additional Wnts may participate. Here, we demonstrate that Wnt11 is a mediator of osteoblast commitment of MSCs, and increases in a surface-roughness dependent manner. Experiments using cells silenced for Wnt11 indicate that cross-talk between Wnt5a and Wnt11 occurs. Wnt11 potentially acts upstream to Wnt5a, increasing Wnt5a expression and factors associated with osteogenesis. Thus, Wnt11 contributes to peri-implant bone formation distal to the implant surface through a heavily regulated signaling cascade of autocrine/paracrine proteins.

Microencapsulated rabbit adipose stem cells initiate tissue regeneration in a rabbit ear defect model.

Cell-based tissue engineering can promote cartilage tissue regeneration, but cell retention in the implant site post-delivery is problematic. Alginate microbeads containing adipose stem cells (ASCs) pretreated with chondrogenic media have been used successfully to regenerate hyaline cartilage in critical size defects in rat xiphoid suggesting that they may be used to treat defects in elastic cartilages such as the ear. To test this, we used microbeads made with low viscosity, high mannuronate medical grade alginate using a high electrostatic potential, and a calcium cross linking solution containing glucose. Microbeads containing rabbit ASCs (rbASCs) were implanted bilaterally in 3 mm critical size midcartilage ear defects of six skeletally mature male New Zealand White rabbits (empty defect; microbeads without cells; microbeads with cells; degradable microbeads with cells; and autograft). Twelve weeks post-implantation, regeneration was assessed by microCT and histology. Microencapsulated rbASCs cultured in chondrogenic media expressed mRNAs for aggrecan, Type II collagen, and Type X collagen. Histologically, empty defects contained fibrous tissue; microbeads without cells were still present in defects and were surrounded by fibrous tissue; nondegradable beads with rbASCs initiated cartilage regeneration; degradable microbeads with cells produced immature bone-like tissue, also demonstrated by microCT; and autografts appeared as normal auricular cartilage but were not fully integrated with the tissue surrounding the defect. Elastin, the hallmark of auricular cartilage, was not evident in the neocartilage. This delivery system offers the potential for regeneration of auricular cartilage, but vascularity of the treatment site and use of factors that induce elastin must be considered.

MicroRNA Contents in Matrix Vesicles Produced by Growth Plate Chondrocytes are Cell Maturation Dependent.

Chondrocytes at different maturation states in the growth plate produce matrix vesicles (MVs), membrane organelles found in the extracellular matrix, with a wide range of contents, such as matrix processing enzymes and receptors for hormones. We have shown that MVs harvested from growth zone (GC) chondrocyte cultures contain abundant small RNAs, including miRNAs. Here, we determined whether RNA also exists in MVs produced by less mature resting zone (RC) chondrocytes and, if so, whether it differs from the RNA in MVs produced by GC cells. Our results showed that RNA, small RNA specifically, was present in RC-MVs, and it was well-protected from RNase by the phospholipid membrane. A group of miRNAs was enriched in RC-MVs compared RC-cells, suggesting that miRNAs are selectively packaged into MVs. High throughput array and RNA sequencing showed that ~39% miRNAs were differentially expressed between RC-MVs and GC-MVs. Individual RT-qPCR also confirmed that miR-122-5p and miR-150-5p were expressed at significantly higher levels in RC-MVs compared to GC-MVs. This study showed that growth plate chondrocytes at different differentiation stages produce different MVs with different miRNA contents, further supporting extracellular vesicle miRNAs play a role as "matrisomes" that mediate the cell-cell communication in cartilage and bone development.

Surface modification of bulk titanium substrates for biomedical applications via low-temperature microwave hydrothermal oxidation.

Micro-to-nanoscale surface topographies of orthopaedic and dental implants can affect fluid wetting and biological response. Nanoscale features can be superimposed on microscale roughness of titanium (Ti) surfaces at high temperatures, resulting in increased osteoblast differentiation. However, high temperatures can compromise mechanical properties of the bulk material. Here, we have developed a novel low-temperature microwave hydrothermal (MWHT) oxidation process for nanomodification of microrough (SLA) Ti surfaces. Nanoscale protuberances (20 -100 nm average diameter) were generated on SLA surfaces via MWHT treatment at 200°C in H2 O, or in aqueous solutions of H2 O2 or NH4 OH, for times ranging from 1 to 40 h. The size, shape, and crystalline content of the nanoprotuberances varied with the solution used and treatment time. The hydrophilicity of all MWHT-modified surfaces was dramatically enhanced. MG63 and normal human osteoblasts (NHOsts) were cultured on MWHT-treated SLA surfaces. While most responses to MWHT-modified surfaces were comparable to those seen on SLA controls, the MWHT-generated nanotopography reduced osteocalcin production by NHOst cells, suggesting that specific nanotopographic characteristics differentially mediate osteoblast phenotypic expression. MWHT processing provides a scalable, low-temperature route for tailoring nanoscale topographies on microroughened titanium implant surfaces with significantly enhanced wetting by water, without degrading the microscale surface structure of such implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 782-796, 2018.

Inhibition of angiogenesis impairs bone healing in an in vivo murine rapid resynostosis model.

Biologics can improve bone formation, but may diffuse away from sites of therapeutic need. We developed a click-chemistry hydrogel that rapidly polymerizes in situ to control delivery of biologics during post-suturectomy resynostosis in 21-day-old male mice. Here, we used this model to determine the role of angiogenesis in post-suturectomy resynostosis and examine whether controlled release of angiogenesis inhibitors could delay bone regeneration. Hydrogels [DB-co-PEG/poly (TEGDMA)-co-(N3-TEGDMA)] were produced containing anti-angiogenic compounds [anti-VEGFA-antibody or hypoxia inducible factor 1α-inhibitor topotecan]. Bioactivity in vitro was assessed by tube length and branching points of endothelial cells in hydrogel-conditioned media. In vivo effects were examined 14 day post-suturectomy, based on the temporal analysis of angiogenic mRNAs during resynostosis following posterior frontal suture removal. MicroCT was used to quantify angiogenesis in contrast-agent-perfused blood vessels and bone defect size in defects receiving hydrogel, anti-VEGFA/hydrogel, or topotecan/hydrogel. Shorter endothelial tube length and less branching were seen in inhibitor-conditioned media (topotecan > AbVEGFA). In vivo, both compounds inhibited angiogenesis compared with hydrogel-only. Anti-VEGFA/hydrogel reduced resynostosis compared with empty defects, but topotecan/hydrogel blocked bone regeneration. We demonstrate that anti-angiogenic compounds can be incorporated into a spontaneously polymerizing hydrogel and remain active over 14 days in vitro and in vivo. Moreover, bone formation can be delayed by inhibiting neovascularization, suggesting possible use as a therapeutic to control resynostosis following suturectomies and potential applications in other conditions where rapid osteogenesis is not desired. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2742-2749, 2017.

Laser Sintered Porous Ti-6Al-4V Implants Stimulate Vertical Bone Growth.

The objective of this study was to examine the ability of 3D implants with trabecular-bone-inspired porosity and micro-/nano-rough surfaces to enhance vertical bone ingrowth. Porous Ti-6Al-4V constructs were fabricated via laser-sintering and processed to obtain micro-/nano-rough surfaces. Male and female human osteoblasts were seeded on constructs to analyze cell morphology and response. Implants were then placed on rat calvaria for 10 weeks to assess vertical bone ingrowth, mechanical stability and osseointegration. All osteoblasts showed higher levels of osteocalcin, osteoprotegerin, vascular endothelial growth factor and bone morphogenetic protein 2 on porous constructs compared to solid laser-sintered controls. Porous implants placed in vivo resulted in an average of 3.1 ± 0.6 mm3 vertical bone growth and osseointegration within implant pores and had significantly higher pull-out strength values than solid implants. New bone formation and pull-out strength was not improved with the addition of demineralized bone matrix putty. Scanning electron images and histological results corroborated vertical bone growth. This study indicates that Ti-6Al-4V implants fabricated by additive manufacturing to have porosity based on trabecular bone and post-build processing to have micro-/nano-surface roughness can support vertical bone growth in vivo, and suggests that these implants may be used clinically to increase osseointegration in challenging patient cases.

Bone Morphogenetic Protein 2 Alters Osteogenesis and Anti-Inflammatory Profiles of Mesenchymal Stem Cells Induced by Microtextured Titanium In Vitro.

OBJECTIVES: Microtextured titanium (Ti) induces osteoblast differentiation of mesenchymal stem cells (MSCs) in the absence of exogenous osteogenic factors; and high-energy surface modifications speed healing of microrough Ti implants. Bone morphogenetic protein 2 (BMP2) is used clinically to improve peri-implant bone formation and osseointegration but can cause inflammation and bone-related complications. In this study, we determined whether BMP2 alters human MSC differentiation, apoptosis, and inflammatory factor production when grown on Ti implants with different surface properties. MATERIALS AND METHODS: Human MSCs were cultured on Ti substrates (smooth [PT], sandblasted acid-etched [SLA], hydrophilic-SLA [modSLA]), or tissue culture polystyrene (TCPS). After 7 days, inflammatory mRNAs were measured by polymerase chain reaction array. In addition, 7-day cultures were treated with exogenous BMP2 and osteogenic differentiation and production of local factors, proinflammatory interleukins, and anti-inflammatory interleukins assessed. Finally, osteogenic markers and interleukins were measured in MSCs cultured for 48 h on BMP2 dip-coated SLA and modSLA surfaces. RESULTS: Expression of interleukins, chemokines, cytokines, and growth factors was affected by surface properties, particularly on modSLA. MSCs on Ti produced fewer resorptive and more osteogenic/anti-inflammatory factors than cells on TCPS. Addition of 100 ng/mL BMP2 not only increased differentiation but also increased proinflammatory and decreased anti-inflammatory/antiresorptive factors. Two hundred nanograms per milliliter BMP2 abolished osteogenesis and dramatically increased pro-osteoclastogenic factors. MSCs cultured on BMP2-dip-coated disks produced similar proinflammatory profiles with inhibited osteogenic differentiation and had increased apoptotic markers at the highest doses. CONCLUSIONS: MSCs underwent osteogenesis and regulated inflammatory cytokines on microtextured Ti. Exogenous BMP2 inhibited MSC differentiation and stimulated a dose-dependent proinflammatory and apoptotic response. Use of BMP2 with microtextured metal implants may increase inflammation and possibly delay bone formation dependent on dose, suggesting that application of BMP2 clinically during implant insertion may need to be reevaluated.

Substrate Stiffness Controls Osteoblastic and Chondrocytic Differentiation of Mesenchymal Stem Cells without Exogenous Stimuli.

Stem cell fate has been linked to the mechanical properties of their underlying substrate, affecting mechanoreceptors and ultimately leading to downstream biological response. Studies have used polymers to mimic the stiffness of extracellular matrix as well as of individual tissues and shown mesenchymal stem cells (MSCs) could be directed along specific lineages. In this study, we examined the role of stiffness in MSC differentiation to two closely related cell phenotypes: osteoblast and chondrocyte. We prepared four methyl acrylate/methyl methacrylate (MA/MMA) polymer surfaces with elastic moduli ranging from 0.1 MPa to 310 MPa by altering monomer concentration. MSCs were cultured in media without exogenous growth factors and their biological responses were compared to committed chondrocytes and osteoblasts. Both chondrogenic and osteogenic markers were elevated when MSCs were grown on substrates with stiffness <10 MPa. Like chondrocytes, MSCs on lower stiffness substrates showed elevated expression of ACAN, SOX9, and COL2 and proteoglycan content; COMP was elevated in MSCs but reduced in chondrocytes. Substrate stiffness altered levels of RUNX2 mRNA, alkaline phosphatase specific activity, osteocalcin, and osteoprotegerin in osteoblasts, decreasing levels on the least stiff substrate. Expression of integrin subunits α1, α2, α5, αv, ß1, and ß3 changed in a stiffness- and cell type-dependent manner. Silencing of integrin subunit beta 1 (ITGB1) in MSCs abolished both osteoblastic and chondrogenic differentiation in response to substrate stiffness. Our results suggest that substrate stiffness is an important mediator of osteoblastic and chondrogenic differentiation, and integrin ß1 plays a pivotal role in this process.

Comparable responses of osteoblast lineage cells to microstructured hydrophilic titanium-zirconium and microstructured hydrophilic titanium.

OBJECTIVES: Although titanium (Ti) is commonly used for dental implants, Ti alloy materials are being developed to improve their physical material properties. Studies indicate that osteoblast differentiation and maturation of human mesenchymal stem cells (MSCs) and normal human osteoblasts (NHOsts) respond to microstructured Ti and titanium-aluminum-vanadium (Ti6Al4V) surfaces in a similar manner. The goal of this study was to determine whether this is the case for osteoblast lineage cells grown on microstructured TiZr surfaces and whether their response is affected by surface nanotexture and hydrophilicity. MATERIALS AND METHODS: Grade 4 Ti and TiZr (13-17% Zr) disks were modified by large grit sand-blasting and acid-etching with storage in saline solution, resulting in a complex microstructured and hydrophilic surface corresponding to the commercially available implants SLActive® and Roxolid® SLActive® (Institut Straumann AG, Basel, Switzerland). The subsequent Ti modSLA and TiZr modSLA surfaces were characterized and osteogenic markers were measured. RESULTS: Evaluation of physical parameters revealed that the fabrication method was capable of inducing a microstructured and hydrophilic surface on both the Ti and TiZr disks. Overall, the surfaces were similar, but differences in nanostructure morphology/density and surface chemistry were detected. On Ti modSLA and TiZr modSLA, osteoblastic differentiation and maturation markers were enhanced in both MSCs and NHOsts, while inflammatory markers decreased compared with TCPS. CONCLUSIONS: These results indicate a similar positive cell response of MSCs and NHOsts when cultured on Ti modSLA and TiZr modSLA. Both surfaces were hydrophilic, indicating the importance of this property to osteoblast lineage cells.

Dental implant surface chemistry and energy alter macrophage activation in vitro.

OBJECTIVES: To determine the effects of dental implant surface chemistry and energy on macrophage activation in vitro. MATERIALS AND METHODS: Disks made from two clinically used implant materials (titanium [Ti], titanium zirconium alloy [TiZr]) were produced with two different surface treatments (sandblast/acid-etch [SLA], hydrophilic-SLA [modSLA]). Surface roughness, energy, and chemistry were characterized. Primary murine macrophages were isolated from 6- to 8-week-old male C57Bl/6 mice and cultured on test surfaces (Ti SLA, TiZr SLA, Ti modSLA, TiZr modSLA) or control tissue culture polystyrene. mRNA was quantified by quantitative polymerase chain reaction after 24 h of culture. Pro- (IL-1ß, IL-6, and TNF-α) and anti-inflammatory (IL-4, IL-10) protein levels were measured by ELISA after 1 or 3 days of culture. RESULTS: Quantitatively, microroughness was similar on all surfaces. Qualitatively, nanostructures were present on modSLA surfaces that were denser on Ti than on TiZr. modSLA surfaces were determined hydrophilic (high-energy surface) while SLA surfaces were hydrophobic (low-energy surface). Cells on high-energy surfaces had higher levels of mRNA from anti-inflammatory markers characteristic of M2 activation compared to cells on low-energy surfaces. This effect was enhanced on the TiZr surfaces when compared to cells on Ti SLA and Ti modSLA. Macrophages cultured on TiZr SLA and modSLA surfaces released more anti-inflammatory cytokines. CONCLUSIONS: The combination of high-energy and altered surface chemistry present on TiZr modSLA was able to influence macrophages to produce the greatest anti-inflammatory microenvironment and reduce extended pro-inflammatory factor release.

Effects of low-frequency ultrasound treatment of titanium surface roughness on osteoblast phenotype and maturation.

OBJECTIVE: Low-frequency ultrasound is widely used in the treatment of chronically infected wounds. To investigate its feasibility as a method for in situ restoration of metal implant surfaces in cases of peri-implantitis, we evaluated how low-frequency ultrasound affected surface properties of and response of human osteoblast-like MG63 cells to titanium (Ti). MATERIAL AND METHODS: Three Ti surfaces [hydrophobic/smooth (pretreatment, PT); hydrophobic/rough (sandblasted/acid-etched, SLA); and hydrophilic/rough (SLA processed and stored hydrophilicity, mSLA)] were subjected to 25 kHz ultrasound for 10 min/cm2 . Substrate roughness, chemical composition, and wettability were analyzed before and after ultrasound application. Osteoblastic maturation of cells on sonicated disks was compared to cells on untreated disks. RESULTS: Ultrasound treatment altered the topography of all surfaces. Contact angles were reduced, and chemical compositions were altered by ultrasound on PT and SLA surfaces. Cell response to sonicated PT was comparable to untreated PT. Alkaline phosphatase was increased on sonicated SLA compared to untreated SLA, whereas DNA, osteocalcin, BMP2, osteoprotegerin, and VEGF-A were unchanged. Cells produced less osteocalcin and BMP2 on sonicated mSLA than on untreated mSLA, but no other parameters were affected. CONCLUSIONS: These results show that low-frequency ultrasound altered Ti surface properties. Osteoblasts were sensitive to the changes induced by ultrasound treatment. The data suggest that the effect is to delay differentiation, but it is unclear whether this delay will prevent osseointegration. These results suggest that low-frequency ultrasound may be useful for treating implant surfaces in situ leading to successful re-osseointegration of implants affected by peri-implantitis.

24R,25-Dihydroxyvitamin D3 Protects against Articular Cartilage Damage following Anterior Cruciate Ligament Transection in Male Rats.

Osteoarthritis (OA) in humans is associated with low circulating 25-hydroxyvitamin D3 [25(OH)D3]. In vitamin D replete rats, radiolabeled 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] accumulates in articular cartilage following injection of [3H]-25(OH)D3. Previously, we showed that 24R,25(OH)2D3 blocks chondrocyte apoptosis via phospholipase D and p53, suggesting a role for 24R,25(OH)2D3 in maintaining cartilage health. We examined the ability of 24R,25(OH)2D3 to prevent degenerative changes in articular cartilage in an OA-like environment and the potential mechanisms involved. In vitro, rat articular chondrocytes were treated with IL-1ß with and without 24R,25(OH)2D3 or 1α,25(OH)2D3. 24R,25(OH)2D3 but not 1α,25(OH)2D3 blocked the effects of IL-1ß in a dose-dependent manner, and its effect was partially mediated through the TGF-ß1 signaling pathway. In vivo, unilateral anterior cruciate ligament transections were performed in immunocompetent rats followed by intra-articular injections of 24R,25(OH)2D3 or vehicle (t = 0, 7, 14, 21 days). Tissues were harvested on day 28. Joints treated with vehicle had changes typical of OA whereas joints treated with 24R,25(OH)2D3 had less articular cartilage damage and levels of inflammatory mediators. These results indicate that 24R,25(OH)2D3 protects against OA, and suggest that it may be a therapeutic approach for preventing trauma-induced osteoarthritis.

Hydrogels derived from cartilage matrices promote induction of human mesenchymal stem cell chondrogenic differentiation.

UNLABELLED: Limited supplies of healthy autologous or allogeneic cartilage sources have inspired a growing interest in xenogeneic cartilage matrices as biological scaffolds for cartilage tissue engineering. The objectives of this study were to determine if shark and pig cartilage extracellular matrix (ECM) hydrogels can stimulate chondrocytic differentiation of mesenchymal stem cells (MSCs) without exogenous growth factors and to determine if the soluble factors retained by these ECM hydrogels are responsible. Human MSCs cultured on hydrogels from shark skull cartilage, pig articular cartilage, and pig auricular cartilage ECM had increased expression of chondrocyte markers and decreased secretion of angiogenic factors VEGF-A and FGF2 in comparison to MSCs cultured on tissue culture polystyrene (TCPS) at one week. MSCs grown on shark ECM gels had decreased type-1 collagen mRNA as compared to all other groups. Degradation products of the cartilage ECM gels and soluble factors released by the matrices increased chondrogenic and decreased angiogenic mRNA levels, indicating that the processed ECM retains biochemically active proteins that can stimulate chondrogenic differentiation. In conclusion, this work supports the use of cartilage matrix-derived hydrogels for chondrogenic differentiation of MSCs and cartilage tissue engineering. Longer-term studies and positive controls will be needed to support these results to definitively demonstrate stimulation of chondrocyte differentiation, and particularly to verify that calcification without endochondral ossification does not occur as it does in shark cartilage. STATEMENT OF SIGNIFICANCE: The objectives of this study were to determine if shark and pig cartilage extracellular matrix (ECM) hydrogels can stimulate chondrocytic differentiation of mesenchymal stem cells (MSCs) without exogenous growth factors and to determine if the soluble factors retained by these ECM hydrogels are responsible for this induction. Sharks are an especially interesting model for cartilage regeneration because their entire skeleton is composed of cartilage and they do not undergo endochondral ossification. Culturing human MSCs on porcine and shark cartilage ECM gels directly, with ECM gel conditioned media, or degradation products increased mRNA levels of chondrogenic factors while decreasing angiogenic factors. These studies indicate that xenogeneic cartilage ECMs have potential as biodegradable scaffolds capable of stimulating chondrogenesis while preventing angiogenesis for regenerative medicine applications and that ECM species selection can yield differential effects.

Characterization of osteoarthritic human knees indicates potential sex differences.

BACKGROUND: The prevalence of osteoarthritis is higher in women than in men in every age group, and overall prevalence increases with advancing age. Sex-specific differences in the properties of osteoarthritic joint tissues may permit the development of sex-specific therapies. Sex hormones regulate cartilage and bone development and homeostasis in a sex-dependent manner. Recent in vitro studies show that the vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] also has sex-specific effects on musculoskeletal cells, suggesting that vitamin D3 metabolites may play a role in osteoarthritis-related sex-specific differences. The purpose of this study was to determine if sex-specific differences exist in synovial fluid and knee tissues isolated from male and female patients with severe knee osteoarthritis. We determined the presence of vitamin D3 metabolites, inflammatory cytokines, growth factors, and matrix metalloproteinases (MMPs) in synovial fluid and assessed responses of articular chondrocytes and subchondral osteoblasts to 17ß-estradiol, dihydrotestosterone, and 1α,25(OH)2D3. METHODS: Samples from knee joints of 10 Caucasian male and 10 Caucasian female patients with advanced osteoarthritis aged 65 to 75 years were obtained from total knee arthroplasty. Vitamin D metabolites, cytokines, MMPs, and growth factors in the synovial fluid were measured. Primary cultures of chondrocytes were isolated from fibrillated articular cartilage adjacent to osteoarthritis lesions and minimally affected cartilage distal to the lesion. Osteoblasts were isolated from the subchondral bone. Expression of receptors for 17ß-estradiol and 1α,25(OH)2D3 was assessed by real-time PCR. Chondrocytes and osteoblasts were treated with 10(-8) M 17ß-estradiol, dihydrotestosterone, or 1α,25(OH)2D3 and effects on gene expression and protein synthesis determined. RESULTS: Histology of the articular cartilage confirmed advanced osteoarthritis. Sex differences were found in synovial fluid levels of vitamin D metabolites, cytokines, and metalloproteinases as well as in the cellular expression of receptors for 17ß-estradiol and 1α,25(OH)2D3. Male cells were more responsive to 1α,25(OH)2D3 and dihydrotestosterone, whereas 17ß-estradiol-affected female cells. CONCLUSIONS: These results demonstrate that there are underlying sex differences in knee tissues affected by osteoarthritis. Our findings do not address osteoarthritis etiology but have implications for different prevention methods and treatments for men and women. Further research is needed to better understand these sex-based differences.

Role of integrin α7ß1 signaling in myoblast differentiation on aligned polydioxanone scaffolds.

UNLABELLED: The aligned structural environment in skeletal muscle is believed to be a crucial component in functional muscle regeneration. Myotube formation is increased on aligned biomaterials, but we do not fully understand the mechanisms that direct this enhanced fusion. Previous studies indicate that the α7 integrin subunit is upregulated during myoblast differentiation, suggesting that signaling via α7ß1 mediates the effect of alignment. To test this hypothesis, we took advantage of an in vitro model using random and aligned polydioxanone (PDO) matrices and C2C12 myoblasts. We measured expression and production of myoblast markers: paired box-7 (Pax7), myogenic differentiation factor-1 (MyoD), myogenin (MyoG), myogenic factor-6 (Myf6), and myosin heavy chain (MyHC). To examine the role of α7ß1 signaling, we measured expression and production of α7, α5, and ß1 and myoblast markers in wild type cells and in cells silenced for α7 and assessed effects of silencing on myogenic differentiation. Downstream signaling via ERK1/2 mitogen activated protein kinase (MAPK) was examined using a specific MEK1/2 inhibitor. Alignment increased mRNAs and protein for early (MyoD) and late (MyoG, MyHC) myoblast markers in comparison to non-aligned matrices, and these levels corresponded with increased α7 protein. α7-silencing reduced MyoG and MyHC protein in cells cultured on tissue culture polystyrene and aligned PDO matrices compared to wild type cells. Inhibition of ERK1/2 blocked effects of alignment. These data suggest that alignment regulates myogenic differentiation via α7ß1 integrin signaling and ERK1/2 mediated gene expression. STATEMENT OF SIGNIFICANCE: Muscle regeneration in severe muscle injuries is complex, requiring a sequence of events to promote healing and not fibrosis. Aligned biomaterials that recapitulate muscle environments hold potential to facilitate regeneration, but it is important to understand cell-substrate signaling to form functional muscle. A critical component of muscle signaling is integrin α7ß1, where mice lacking α7 exhibit a dystrophic phenotype and impaired regeneration. Here, we report the role of α7ß1 signaling in myoblast differentiation on aligned biomaterials. α7-silenced myoblasts were found to regulate myogenic differentiation and demonstrate defective fusion. Our data shows reduced levels of myogenin and myosin heavy chain protein, while MyoD remains unchanged. These results support the hypothesis that α7ß1 signaling plays a role in substrate-dependent tissue engineering strategies.

Differential spatial regulation of BMP molecules is associated with single-suture craniosynostosis.

OBJECTIVE The aim of this study was to examine messenger RNA (mRNA) levels of bone morphogenetic protein (BMP) ligands, receptors, and soluble inhibitors in cells isolated from single-suture synostoses from fused coronal, metopic, sagittal, and lambdoid sutures. METHODS Cells were isolated from bone collected from patients undergoing craniotomies at Children's Healthcare of Atlanta. Real-time polymerase chain reaction was used to examine mRNA levels in cells isolated from fused sutures or patent sutures in comparison with levels in normal bone from the same patient. RESULTS Cells isolated from fused sutures in cases of sagittal and coronal synostosis highly expressed BMP2, while cells isolated from fused metopic or lambdoid synostosis expressed high BMP4. Noggin, a BMP inhibitor, was lower in fused sutures and had high expression in patent sutures. CONCLUSIONS These results suggest that BMPs and inhibitors play a significant role in the regulation of suture fusion as well in the maintenance of patency in the normal suture.