van Wijnen, Andre J

Objective This proof-of-concept study investigated an improved cell-based injection therapy combining mesenchymal stem cells (MSCs) and meniscus cells (MCs) to support superior meniscus allograft repopulation and early revival compared to injecting MSCs alone. Design In this controlled laboratory study, frozen meniscus allograft samples were injected vertically with a cell suspension containing different ratios of MSCs and MCs or control (lactated ringers) and cultured for 28 days. Samples were analyzed weekly for cell viability, migration, and metabolism using histological and biochemical assays. Tissue medium was analyzed for matrix metalloproteinase (MMP) expression using zymography. Results Cellular repopulation of frozen allografts injected with different cell suspensions was validated by immunohistochemistry. Significant higher DNA content was evidenced in grafts treated with suspensions of MCs or MC:MSC (1:4 ratio). Cell metabolic activity was significantly different between all treated groups and control group after 1 week. Allografts injected with MCs showed significantly more cell proliferation than injections with MSCs. MMP2 activity was detected in medium of all grafts cellularized with MCs with or without MSCs. Scanning electron microscopy (SEM) analysis showed resolution of the needle puncture, but not in the control group. Cell labeling of MCs upon injection of mixed MC:MSC suspensions revealed a gradual increase in the cell ratio. Conclusions The findings of this study establish that injection of MCs with or without MSCs enhances the cellularity of meniscus allograft to support early graft revival and remodeling.

AbstractArthrofibrosis, which causes joint motion restrictions, is a common complication following total knee arthroplasty (TKA). Key features associated with arthrofibrosis include myofibroblast activation, knee stiffness, and excessive scar tissue formation. We previously demonstrated that adiponectin levels are suppressed within the knee tissues of patients affected by arthrofibrosis and showed that AdipoRon, an adiponectin receptor agonist, exhibited anti‐fibrotic properties in human mesenchymal stem cells. In this study, the therapeutic potential of AdipoRon was evaluated on TGFβ1‐mediated myofibroblast differentiation of primary human knee fibroblasts and in a mouse model of knee stiffness. Picrosirius red staining revealed that AdipoRon reduced TGFβ1‐induced collagen deposition in primary knee fibroblasts derived from patients undergoing primary TKA and revision TKA for arthrofibrosis. AdipoRon also reduced mRNA and protein levels of ACTA2, a key myofibroblast marker. RNA‐seq analysis corroborated the anti‐myofibrogenic effects of AdipoRon. In our knee stiffness mouse model, 6 weeks of knee immobilization, to induce a knee contracture, in conjunction with daily vehicle (DMSO) or AdipoRon (1, 5, and 25 mg/kg) via intraperitoneal injections were well tolerated based on animal behavior and weight measurements. Biomechanical testing demonstrated that passive extension angles (PEAs) of experimental knees were similar between vehicle and AdipoRon treatment groups in mice evaluated immediately following immobilization. Interestingly, relative to vehicle‐treated mice, 5 mg/kg AdipoRon therapy improved the PEA of the experimental knees in mice that underwent 4 weeks of knee remobilization following the immobilization and therapy. Together, these studies revealed that AdipoRon may be an effective therapeutic modality for arthrofibrosis.

ABSTRACT Vitamin C (VitC) is essential for bone health, and low VitC serum levels increase the risk for skeletal fractures. If and how VitC affects bone mineralization is unclear. Using micro-computed tomography (μCT), histologic staining, as well as quantitative backscattered electron imaging (qBEI), we assessed the effects of VitC on femoral structure and microarchitecture, bone formation, and bone mineralization density distribution (BMDD) in the VitC incompetent Gulo−/− mouse model and wild-type mice. In particular, VitC-supplemented, 20-week-old mice were compared with age-matched counterparts where dietary VitC intake was excluded from week 15. VitC depletion in Gulo−/− mice severely reduced cortical thickness of the diaphyseal shaft and bone volume around the growth plate (eg, bone volume of the primary spongiosa −43%, p < 0.001). Loss of VitC also diminished the amount of newly formed bone tissue as visualized by histology and calcein labeling of the active mineralization front. BMDD analysis revealed a shift to higher calcium concentrations upon VitC supplementation, including higher average (~10% increase in female VitC deficient mice, p < 0.001) and peak calcium concentrations in the epiphyseal and metaphyseal spongiosa. These findings suggest higher bone tissue age. Importantly, loss of VitC had significantly more pronounced effects in female mice, indicating a higher sensitivity of their skeleton to VitC deficiency. Our results reveal that VitC plays a key role in bone formation rate, which directly affects mineralization. We propose that low VitC levels may contribute to the higher prevalence of bone-degenerative diseases in females and suggest leveraging this vitamin against these conditions. © 2023 American Society for Bone and Mineral Research (ASBMR). Abstract Vitamin C deficiency disrupts bone microarchitecture, as well as bone formation and mineralization in Gulo−/− mice, which emphasizes the importance of this micronutrient for the skeleton. Importantly, the female skeleton is significantly more sensitive to vitamin C deficiency than the male skeleton, indicating sex-specific differences in vitamin C requirements for bone.

Abstract Background Chronic injuries to the scapholunate ligament (SLIL) alter carpal kinematics and may progress to early degenerative osteoarthritis. To date, there is no consensus for the best method for SLIL reconstruction. This study aims to assess the use of growth factors (bone morphogenetic protein [BMP]2 and growth and differentiation factor 5 [GDF5]) for compartmentalized regeneration of bone and ligament in this multiphasic scaffold in a rabbit knee model. Case Description A total of 100 µg of BMP2 and 30 µg of GDF5 were encapsulated into a heparinized gelatin–hyaluronic acid hydrogel and loaded into the appropriate compartment of the multiphasic scaffold. The multiphasic scaffold was implanted to replace the native rabbit medial collateral ligament (n = 16). The rabbits were randomly assigned to two different treatment groups. The first group was immobilized postoperatively with the knee pinned in flexion with K-wires for 4 weeks (n = 8) prior to sacrifice. The second group was immobilized for 4 weeks, had the K-wires removed followed by a further 4 weeks of mobilization prior to sample harvesting. Literature Review Heterotopic ossification as early as 4 weeks was noted on gross dissection and confirmed by microcomputed tomography and histological staining. This analysis revealed formation of a bony bridge located within and over the ligament compartment in the intra-articular region. Biomechanical testing showed increased ultimate force of the ligament compartment at 4 weeks postimplantation consistent with the presence of bone formation and higher numbers of scaffold failures at the bone–tendon junction. This study has demonstrated that the addition of BMP2 and GDF5 in the bone–ligament–bone (BLB) scaffold resulted in heterotopic bone formation and failure of the ligament compartment. Clinical Relevance The implantation of a three-dimensional-printed BLB scaffold alone demonstrated superior biomechanical and histological results, and further investigation is needed as a possible clinical reconstruction for the SLIL.

AbstractArthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition. Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra‐terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA‐seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT‐qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFβ1‐induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleled by siRNA‐mediated depletion of BRD4. Together, these data reveal that BRD4‐mediated epigenetic events support TGFβ1‐mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis.

AbstractArboviruses target bone forming osteoblasts and perturb bone remodeling via paracrine factors. We previously reported that Zika virus (ZIKV) infection of early‐stage human mesenchymal stromal cells (MSCs) inhibited the osteogenic lineage commitment of MSCs. To understand the physiological interplay between bone development and ZIKV pathogenesis, we employed a primary in vitro model to examine the biological responses of MSCs to ZIKV infection at different stages of osteogenesis. Precommitted MSCs were infected at the late stage of osteogenic stimulation (Day 7) with ZIKV (multiplicity of infection of 5). We observe that MSCs infected at the late stage of differentiation are highly susceptible to ZIKV infection similar to previous observations with early stage infected MSCs (Day 0). However, in contrast to ZIKV infection at the early stage of differentiation, infection at a later stage significantly elevates the key osteogenic markers and calcium content. Comparative RNA sequencing (RNA‐seq) of early and late stage infected MSCs reveals that ZIKV infection alters the mRNA transcriptome during osteogenic induction of MSCs (1251 genes). ZIKV infection provokes a robust antiviral response at both stages of osteogenic differentiation as reflected by the upregulation of interferon responsive genes (n > 140). ZIKV infection enhances the expression of immune‐related genes in early stage MSCs while increasing cell cycle genes in late stage MSCs. Remarkably, ZIKA infection in early stage MSCs also activates lipid metabolism‐related pathways. In conclusion, ZIKV infection has differentiation stage‐dependent effects on MSCs and this mechanistic understanding may permit the development of new therapeutic or preventative measures for bone‐related effects of ZIKV infection.

Dental occlusion requires harmonious development of teeth, jaws, and other elements of the craniofacial complex, which are regulated by environmental and genetic factors. We performed the first genome-wide association study (GWAS) on dental development (DD) using the Demirjian radiographic method. Radiographic assessments from participants of the Generation R Study (primary study population, N 1 = 2,793; mean age of 9.8 y) were correlated with ~30 million genetic variants while adjusting for age, sex, and genomic principal components (proxy for population stratification). Variants associated with DD at genome-wide significant level ( P < 5 × 10 −8 ) mapped to 16q12.2 ( IRX5) (lead variant rs3922616, B = 0.16; P = 2.2 × 10 −8 ). We used Fisher’s combined probability tests weighted by sample size to perform a meta-analysis ( N = 14,805) combining radiographic DD at a mean age of 9.8 y from Generation R with data from a previous GWAS ( N 2 = 12,012) on number of teeth (NT) in infants used as proxy of DD at a mean age of 9.8 y (including the ALSPAC and NFBC1966). This GWAS meta-analysis revealed 3 novel loci mapping to 7p15.3 ( IGF2BP3: P = 3.2 × 10 −8 ), 14q13.3 ( PAX9: P = 1.9 × 10 −8 ), and 16q12.2 ( IRX5: P = 1.2 × 10 −9 ) and validated 8 previously reported NT loci. A polygenic allele score constructed from these 11 loci was associated with radiographic DD in an independent Generation R set of children ( N = 703; B = 0.05, P = 0.004). Furthermore, profiling of the identified genes across an atlas of murine and human stem cells observed expression in the cells involved in the formation of bone and/or dental tissues (>0.3 frequency per kilobase of transcript per million mapped reads), likely reflecting functional specialization. Our findings provide biological insight into the polygenic architecture of the pediatric dental maturation process.

Abstract Atherosclerotic renal artery stenosis (ARAS) is associated with irreversible parenchymal renal disease and regenerative stem cell therapies may improve renal outcomes. Hypoxia preconditioning (HPC) may improve the regenerative functions of adipose tissue-derived mesenchymal stem cells (AMSC) by affecting DNA 5-hydroxymethylcytosine (5hmC) marks in angiogenic genes. Here, we investigated using a porcine ARAS model, whether growth of ARAS AMSCs in hypoxia (Hx) versus normoxia (Nx) would enhance renal tissue repair, and comprehensively analyze how HPC modifies DNA hydroxymethylation compared to untreated ARAS and healthy/normal pigs (n=5 each). ARAS pigs exhibited elevated serum cholesterol, serum creatinine and renal artery stenosis, with a concomitant decrease in renal blood flow (RBF) and increased blood pressure (BP) compared to healthy pigs. Renal artery injection of either autologous Nx or Hx AMSCs improved diastolic BP, reduced kidney tissue fibrosis, and inflammation (CD3+ T-cells) in ARAS pigs. In addition, renal medullary hypoxia significantly lowered with Nx but not Hx AMSC treatment. Mechanistically, levels of epigenetic 5hmC marks (which reflect gene activation) estimated using DNA immunoprecipitation technique were elevated in profibrotic and inflammatory genes in ARAS compared with normal AMSCs. HPC significantly reduced 5hmC levels in cholesterol biosynthesis and oxidative stress response pathways in ARAS AMSCs. Thus, autologous AMSCs improve key renovascular parameters and inflammation in ARAS pigs, with HPC mitigating pathological molecular effects on inflammatory and profibrotic genes which may play a role in augmenting regenerative capacity of AMSCs.