Identification of a fundamental cryoinjury mechanism in MSCs and its mitigation through cell-cycle synchronization prior to freezing.

Clinical development of cellular therapies, including mesenchymal stem/stromal cell (MSC) treatments, has been hindered by ineffective cryopreservation methods that result in substantial loss of post-thaw cell viability and function. Proposed solutions to generate high potency MSC for clinical testing include priming cells with potent cytokines such as interferon gamma (IFNγ) prior to cryopreservation, which has been shown to enhance post-thaw function, or briefly culturing to allow recovery from cryopreservation injury prior to administering to patients. However, both solutions have disadvantages: cryorecovery increases the complexity of manufacturing and distribution logistics, while the pleiotropic effects of IFNγ may have uncharacterized and unintended consequences on MSC function. To determine specific cellular functions impacted by cryoinjury, we first evaluated cell cycle status. It was discovered that S phase MSC are exquisitely sensitive to cryoinjury, demonstrating heightened levels of delayed apoptosis post-thaw and reduced immunomodulatory function. Blocking cell cycle progression at G0/G1 by growth factor deprivation (commonly known as serum starvation) greatly reduced post-thaw dysfunction of MSC by preventing apoptosis induced by double-stranded breaks in labile replicating DNA that form during the cryopreservation and thawing processes. Viability, clonal growth and T cell suppression function were preserved at pre-cryopreservation levels and were no different than cells prior to freezing or frozen after priming with IFNγ. Thus, we have developed a robust and effective strategy to enhance post-thaw recovery of therapeutic MSC.

Associations between collagen X biomarker and linear growth velocity in a pediatric chronic kidney disease cohort.

BACKGROUND: Collagen X biomarker (CXM) is a novel biomarker of linear growth velocity. We investigated whether CXM correlated with measured growth velocity in children with impaired kidney function. METHODS: We used data from children aged 2 through 16 years old enrolled in the Chronic Kidney Disease in Children (CKiD) study. We assessed the association between CXM level and growth velocity based on height measurements obtained at study visits using linear regression models constructed separately by sex, with and without adjustment for CKD covariates. Linear mixed-effects models were used to capture the between-individual and within-individual CXM changes over time associated with concomitant changes in growth velocity from baseline through follow-up. RESULTS: A total of 967 serum samples from 209 participants were assayed for CXM. CXM correlated more strongly in females compared to male participants. After adjustment for growth velocity and CKD covariates, only proteinuria in male participants affected CXM levels. Finally, we quantified the between- and within-participant associations between CXM level and growth velocity. A between-participant increase of 24% and 15% in CXM level in females and males, respectively, correlated with a 1 cm/year higher growth velocity. Within an individual participant, on average, 28% and 13% increases in CXM values in females and males, respectively, correlated with a 1 cm/year change in measured growth. CONCLUSIONS: CXM measurement is potentially a valuable aid for monitoring growth in pediatric CKD. However, future research, including studies of CXM metabolism, is needed to clarify whether CXM can be a surrogate of growth in children with CKD. A higher resolution version of the Graphical abstract is available as Supplementary information.

Identification of potential non-invasive biomarkers in diastrophic dysplasia.

Diastrophic dysplasia (DTD) is a recessive chondrodysplasia caused by pathogenic variants in the SLC26A2 gene encoding for a cell membrane sulfate/chloride antiporter crucial for sulfate uptake and glycosaminoglycan (GAG) sulfation. Research on a DTD animal model has suggested possible pharmacological treatment approaches. In view of future clinical trials, the identification of non-invasive biomarkers is crucial to assess the efficacy of treatments. Urinary GAG composition has been analyzed in several metabolic disorders including mucopolysaccharidoses. Moreover, the N-terminal fragment of collagen X, known as collagen X marker (CXM), is considered a real-time marker of endochondral ossification and growth velocity and was studied in individuals with achondroplasia and osteogenesis imperfecta. In this work, urinary GAG sulfation and blood CXM levels were investigated as potential biomarkers for individuals affected by DTD. Chondroitin sulfate disaccharide analysis was performed on GAGs isolated from urine by HPLC after GAG digestion with chondroitinase ABC and ACII, while CXM was assessed in dried blood spots. Results from DTD patients were compared with an age-matched control population. Undersulfation of urinary GAGs was observed in DTD patients with some relationship to the clinical severity and underlying SLC26A2 variants. Lower than normal CXM levels were observed in most patients, even if the marker did not show a clear pattern in our small patient cohort because CXM values are highly dependent on age, gender and growth velocity. In summary, both non-invasive biomarkers are promising assays targeting various aspects of the disorder including overall metabolism of sulfated GAGs and endochondral ossification.

Accelerating cryoprotectant delivery using vacuum infiltration.

The ability to cryopreserve bone marrow within the vertebral body (VB) would offer significant clinical and research benefits. However, cryopreservation of large structures, such as VBs, is challenging due to mass transport limitations that prevent the effective delivery of cryoprotectants into the tissue. To overcome this challenge, we examined the potential of vacuum infiltration, along with carbonation, to increase the penetration of cryoprotectants. In particular, we hypothesized that initial exposure to high-pressure carbon dioxide gas would introduce bubbles into the tissue and that subsequent vacuum cycling would cause expansion and contraction of the bubbles, thus enhancing the transport of cryoprotectant into the tissue. Experiments were carried out using colored dye and agarose gel as a model revealing that carbonation and vacuum cycling result in a 14% increase in dye penetration compared to the atmospheric controls. Experiments were also carried out by exposing VBs isolated from human vertebrae to 40% (v/v) DMSO solution. CT imaging showed the presence of gas bubbles within the tissue pores for carbonated VBs as well as control VBs. Vacuum cycling reduced the bubble volume by more than 50%, most likely resulting in replacement of this volume with DMSO solution. However, we were unable to detect a statistically significant increase in DMSO concentration within the VBs using CT imaging. This research suggests that there may be a modest benefit to carbonation and vacuum cycling for introduction of cryoprotectants into larger structures, like VBs.

Fast, non-eccentrically loaded exercise worsens tendinopathic healing responses in a murine model.

OBJECTIVE: To advance the understanding of how alterations in exercise speed and grade (flat vs 17° incline or decline) affect the quality of tendon healing, and to determine if a biomarker relationship exists between serum levels of a ColX breakdown product (CXM) and animals exposed to treadmill running protocols. ANIMALS: 35 male mice (C57BL/6J), 8 weeks of age. PROCEDURES: Mice were preconditioned on a treadmill for 14 days. Tendinopathy was then induced by 2 intra-tendinous TGFß1 injections followed by randomization into 7 exercise groups. Exercise capacity and objective gait analysis were measured weekly. Mice were euthanized and histopathologic analysis and evaluation of serum CXM levels were performed. Statistics were conducted using a 2-way ANOVA (exercise capacity), Mixed Effects Model (gait analysis, effect of preconditioning), and 1-way ANOVA (gait analysis, the effect of injury, and rehabilitation normalized to baseline; CXM serum analysis), all with Tukey post hoc tests and significance set to P < .05. RESULTS: Exercise at a fast-flat speed demonstrated inferior tendinopathic healing at the cellular level and impaired stance braking abilities, which were compensated for by increased propulsion. Mice exposed to exercise (at any speed or grade) demonstrated higher systemic levels of CXM than those that were cage rested. However, no ColX immunostaining was observed in the Achilles tendon or calcaneal insertion. CLINICAL RELEVANCE: Exercise at a fast speed and in absence of eccentric loading components (incline or decline) demonstrated inferior tendinopathic healing at the cellular level and impaired braking abilities that were compensated for by increased propulsion.

Characterization and Function of Cryopreserved Bone Marrow from Deceased Organ Donors: A Potential Viable Alternative Graft Source.

Despite the readily available graft sources for allogeneic hematopoietic cell transplantation (alloHCT), a significant unmet need remains in the timely provision of suitable unrelated donor grafts. This shortage is related to the rarity of certain HLA alleles in the donor pool, nonclearance of donors owing to infectious disease or general health status, and prolonged graft procurement and processing times. An alternative hematopoietic progenitor cell (HPC) graft source obtained from the vertebral bodies (VBs) of deceased organ donors could alleviate many of the obstacles associated with using grafts from healthy living donors or umbilical cord blood (UCB). Deceased organ donor-derived bone marrow (BM) can be preemptively screened, cryogenically banked for on-demand use, and made available in adequate cell doses for HCT. We have developed a good manufacturing practice (GMP)-compliant process to recover and cryogenically bank VB-derived HPCs from deceased organ donor (OD) BM. Here we present results from an analysis of HPCs from BM obtained from 250 deceased donors to identify any substantial difference in composition or quality compared with HPCs from BM aspirated from the iliac crests of healthy living donors. BM from deceased donor VBs was processed in a central GMP facility and packaged for cryopreservation in 5% DMSO/2.5% human serum albumin. BM aspirated from living donor iliac crests was obtained and used for comparison. A portion of each specimen was analyzed before and after cryopreservation by flow cytometry and colony-forming unit potential. Bone marrow chimerism potential was assessed in irradiated immunocompromised NSG mice. Analysis of variance with Bonferroni correction for multiple comparisons was used to determine how cryopreservation affects BM cells and to evaluate indicators of successful engraftment of BM cells into irradiated murine models. The t test (with 95% confidence intervals [CIs]) was used to compare cells from deceased donors and living donors. A final dataset of complete clinical and matched laboratory data from 226 cryopreserved samples was used in linear regressions to predict outcomes of BM HPC processing. When compared before and after cryopreservation, OD-derived BM HPCs were found to be stable, with CD34+ cells maintaining high viability and function after thawing. The yield from a single donor is sufficient for transplantation of an average of 1.6 patients (range, 1.2 to 7.5). CD34+ cells from OD-derived HPCs from BM productively engrafted sublethally irradiated immunocompromised mouse BM (>44% and >67% chimerism at 8 and 16 weeks, respectively). Flow cytometry and secondary transplantation confirmed that OD HPCs from BM is composed of long-term engrafting CD34+CD38-CD45RA-CD90+CD49f+ HSCs. Linear regression identified no meaningful predictive associations between selected donor-related characteristics and OD BM HPC quality or yield. Collectively, these data demonstrate that cryopreserved BM HPCs from deceased organ donors is potent and functionally equivalent to living donor BM HPCs and is a viable on-demand graft source for clinical HCT. Prospective clinical trials will soon commence in collaboration with the Center for International Blood and Marrow Research to assess the feasibility, safety, and efficacy of Ossium HPCs from BM (ClinicalTrials.gov identifier NCT05068401).

IL-1Ra gene transfer potentiates BMP2-mediated bone healing by redirecting osteogenesis toward endochondral ossification.

An estimated 100,000 patients each year in the United States suffer severe disability from bone defects that fail to heal, a condition where bone-regenerative therapies could provide substantial clinical benefits. Although recombinant human bone morphogenetic protein-2 (rhBMP2) is an osteogenic growth factor that is clinically approved for this purpose, it is only effective when used at exceedingly high doses that incur substantial costs, induce severe inflammation, produce adverse side effects, and form morphologically abnormal bone. Using a validated rat femoral segmental defect model, we show that bone formed in response to clinically relevant doses of rhBMP2 is accompanied by elevated expression of interleukin-1 (IL-1). Local delivery of cDNA encoding the IL-1 receptor antagonist (IL-1Ra) achieved bridging of segmental, critical size defects in bone with a 90% lower dose of rhBMP2. Unlike use of high-dose rhBMP2, bone formation in the presence of IL-1Ra occurred via the native process of endochondral ossification, resulting in improved quality without sacrificing the mechanical properties of the regenerated bone. Our results demonstrate that local immunomodulation may permit effective use of growth factors at lower doses to recapitulate more precisely the native biology of healing, leading to higher-quality tissue regeneration.

Physioxic Culture of Chondrogenic Cells.

Cartilage resides under a low oxygen tension within articulating joints. The oxygen tension within cartilage of the knee joint has been measured to be between 2% and 5% oxygen. Although the literature has historically termed this level of oxygen as hypoxia, particularly when doing experiments in vitro in this range, this is actually the physiological oxygen tension experienced in vivo and is more accurately termed physioxia. In general, culture of chondrogenic cells under physioxia has demonstrated a donor-dependent beneficial effect on chondrogenesis, with an upregulation in cartilage genes (SOX9, COL2A1, ACAN) and matrix deposition (sulfated glycosaminoglycans (sGAGs), collagen II). Physioxia also reduces the expression of hypertrophic markers (COL10A1, MMP13). This chapter will outline the methods for the expansion and differentiation of chondrogenic cells under physioxia using oxygen-controlled incubators and glove box environments, with the typical assays used for qualitative and quantitative assessment of chondrogenesis.

Semaphorin 3A-Neuropilin-1 Signaling Modulates MMP13 Expression in Human Osteoarthritic Chondrocytes.

Osteoarthritis (OA) is a complex disorder of diarthrodial joints caused by multiple risk factors and is characterized by articular cartilage destruction as well as changes in other articular tissues. Semaphorin 3A (Sema3A), known to be a chemo-repellent for sensory nerve fibers, has recently been implicated in cartilage OA pathophysiology. We demonstrated that the expression of SEMA3A and its receptor neuropilin-1 (NRP1) are synchronously upregulated in chondrocytes isolated from knee cartilage of OA patients compared to non-OA control chondrocytes. In addition, we observed that during in vitro passaging of OA chondrocytes, the Nrp-1 level increases, whereas the Sema3A level decreases. In this study, we aimed to uncover how Sema3A-Nrp-1 signaling affects metabolism and viability of OA chondrocytes via siRNA-mediated inhibition of Nrp-1 expression. We observed a decreased proliferation rate and an increase in adhesion and senescence after Nrp-1 silencing. Moreover, MMP13 gene expression was reduced by approximately 75% in NRP1 knockdown OA chondrocytes, whereas MMP13 expression was induced by Sema3A treatment in control (nt siRNA) OA chondrocytes, accompanied by an impaired AKT phosphorylation. These findings suggest a potential catabolic function of Sema3A signaling in OA chondrocytes by inducing MMP13 expression and by compromising pro-survival AKT activation. We propose that targeting the Sema3A-Nrp-1 signaling axis might be an opportunity to interfere with OA pathogenesis and progression.

Evaluating Thera-101 as a Low-Volume Resuscitation Fluid in a Model of Polytrauma.

Traumatic brain injury (TBI) and hemorrhage remain challenging to treat in austere conditions. Developing a therapeutic to mitigate the associated pathophysiology is critical to meet this treatment gap, especially as these injuries and associated high mortality are possibly preventable. Here, Thera-101 (T-101) was evaluated as low-volume resuscitative fluid in a rat model of TBI and hemorrhage. The therapeutic, T-101, is uniquely situated as a TBI and hemorrhage intervention. It contains a cocktail of proteins and microvesicles from the secretome of adipose-derived mesenchymal stromal cells that can act on repair and regenerative mechanisms associated with poly-trauma. T-101 efficacy was determined at 4, 24, 48, and 72 h post-injury by evaluating blood chemistry, inflammatory chemo/cytokines, histology, and diffusion tensor imaging. Blood chemistry indicated that T-101 reduced the markers of liver damage to Sham levels while the levels remained elevated with the control (saline) resuscitative fluid. Histology supports the potential protective effects of T-101 on the kidneys. Diffusion tensor imaging showed that the injury caused the most damage to the corpus callosum and the fimbria. Immunohistochemistry suggests that T-101 may mitigate astrocyte activation at 72 h. Together, these data suggest that T-101 may serve as a potential field deployable low-volume resuscitation therapeutic.

Collagen X Marker Levels are Decreased in Individuals with Achondroplasia.

Collagen X marker (CXM) is a degradation fragment of collagen type X. It is a real-time biomarker of height velocity with established norms. Plasma C-type natriuretic peptide (CNP) and NTproCNP levels have also been found to correlate with growth velocity in the general population and are elevated in individuals with achondroplasia compared with age- and sex-matched controls. Collagen X marker levels in people with fibroblast growth factor receptor 3 (FGFR3)-opathies have never been systematically measured. The objective of this study was to measure CXM in a population of dwarfism caused by FGFR3-opathies. Using the same cohort in which CNP and NTproCNP levels were previously measured, archived serum aliquots from 63 children with achondroplasia, six with hypochondroplasia, and two with thanatophoric dysplasia had CXM concentrations measured. Results were plotted against age- and sex-specific norms, and standard deviation scores were plotted for comparison between clinical diagnoses. CXM levels were significantly decreased (p < 0.0001) in children with achondroplasia compared with age- and sex-matched controls. Temporal patterns of change in CXM levels were sex-dependent. As the FGFR3 pathway was more constitutively active, CXM levels decreased. New tools are emerging to study impact of skeletal dysplasia on growth plate regulation and function.

Collagen X Longitudinal Fracture Biomarker Suggests Staged Fixation in Tibial Plateau Fractures Delays Rate of Endochondral Repair.

OBJECTIVES: To use a novel, validated bioassay to monitor serum concentrations of a breakdown product of collagen X in a prospective longitudinal study of patients sustaining isolated tibial plateau fractures. Collagen X is the hallmark extracellular matrix protein present during conversion of soft, cartilaginous callus to bone during endochondral repair. Previous preclinical and clinical studies demonstrated a distinct peak in collagen X biomarker (CXM) bioassay levels after long bone fractures. SETTING: Level 1 academic trauma facility. PATIENTS/PARTICIPANTS: Thirty-six patients; isolated tibial plateau fractures. INTERVENTION: (3) Closed treatment, ex-fix (temporizing/definitive), and open reduction internal fixation. MAIN OUTCOME MEASUREMENTS: Collagen X serum biomarker levels (CXM bioassay). RESULTS: Twenty-two men and 14 women (average age: 46.3 y; 22.6-73.4, SD 13.3) enrolled (16 unicondylar and 20 bicondylar fractures). Twenty-five patients (72.2%) were treated operatively, including 12 (33.3%) provisionally or definitively treated by ex-fix. No difference was found in peak CXM values between sexes or age. Patients demonstrated peak expression near 1000 pg/mL (average: male-986.5 pg/mL, SD 369; female-953.2 pg/mL, SD 576). There was no difference in peak CXM by treatment protocol, external fixator use, or fracture severity (Schatzker). Patients treated with external fixation (P = 0.05) or staged open reduction internal fixation (P = 0.046) critically demonstrated delayed peaks. CONCLUSIONS: Pilot analysis demonstrates a strong CXM peak after fractures commensurate with previous preclinical and clinical studies, which was delayed with staged fixation. This may represent the consequence of delayed construct loading. Further validation requires larger cohorts and long-term follow-up. Collagen X may provide an opportunity to support prospective interventional studies testing novel orthobiologics or fixation techniques. LEVEL OF EVIDENCE: Level II, prospective clinical observational study.

Surface topography as a tool to detect early changes in a posttraumatic equine model of osteoarthritis.

The equine model of posttraumatic osteoarthritis (OA) mimics certain aspects of the naturally occurring disease, both in horses and humans. The objective of this study was to assess articular cartilage degeneration in a posttraumatic OA model using the established macroscopic and microscopic scoring systems and compare them with a novel surface topography analysis. OA was induced in the carpal joint of 15 (n = 15) mixed breed horses. Surface changes on the articular cartilage were characterized using osteochondral blocks from the third carpal bone (C3) and radial carpal bone using surface topography, standard histological grading, and gross evaluation of the joints. Significant differences were observed between OA and non-OA joints for gross evaluation scores. Microscopic scores of hematoxylin and eosin and Safranin O and Fast Green-stained sections demonstrated no differences between OA and non-OA joints. However, articular cartilage from the induced OA joint had significantly greater surface topography measurements compared with the sham treatment group, consistent with the changes seen on gross evaluation of joints. No significant correlations were noted between surface roughness measurements, histological assessment, and gross evaluation scores. The results suggest that surface topography analysis may provide a reliable objective approach to assess early changes in the cartilage surface in OA.

Substance P and Alpha-Calcitonin Gene-Related Peptide Differentially Affect Human Osteoarthritic and Healthy Chondrocytes.

Osteoarthritis (OA) is a degenerative joint disease that not only causes cartilage loss but also structural damage in all joint tissues. Joints are innervated by alpha-calcitonin gene-related peptide (αCGRP) and substance P (SP)-positive sensory nerve fibers. Alteration of sensory joint innervation could be partly responsible for degenerative changes in joints that contribute to the development of OA. Therefore, our aim was to analyze and compare the molecular effects of SP and αCGRP on the metabolism of articular chondrocytes from OA patients and non-OA cartilage donors. We treated the cells with SP or αCGRP and analysed the influence of these neuropeptides on chondrocyte metabolism and modulation of signaling pathways. In chondrocytes from healthy cartilage, SP had minimal effects compared with its effects on OA chondrocytes, where it induced inflammatory mediators, inhibited chondrogenic markers and promoted apoptosis and senescence. Treatment with αCGRP also increased apoptosis and senescence and reduced chondrogenic marker expression in OA chondrocytes, but stimulated an anabolic and protective response in healthy chondrocytes. The catabolic influence of SP and αCGRP might be due to activation of ERK signaling that could be counteracted by an increased cAMP response. We suggest that a switch between the G-subunits of the corresponding receptors after binding their ligands SP or αCGRP plays a central role in mediating the observed effects of sensory neuropeptides on chondrocytes.

Curcumin-primed human BMSC-derived extracellular vesicles reverse IL-1ß-induced catabolic responses of OA chondrocytes by upregulating miR-126-3p.

BACKGROUND: Curcumin has anti-inflammatory effects and qualifies as a potential candidate for the treatment of osteoarthritis (OA). However, curcumin has limited bioavailability. Extracellular vesicles (EVs) are released by multiple cell types and act as molecule carrier during intercellular communication. We assume that EVs can maintain bioavailability and stability of curcumin after encapsulation. Here, we evaluated modulatory effects of curcumin-primed human (h)BMSC-derived EVs (Cur-EVs) on IL-1ß stimulated human osteoarthritic chondrocytes (OA-CH). METHODS: CellTiter-Blue Viability- (CTB), Caspase 3/7-, and live/dead assays were used to determine range of cytotoxic curcumin concentrations for hBMSC and OA-CH. Cur-EVs and control EVs were harvested from cell culture supernatants of hBMSC by ultracentrifugation. Western blotting (WB), transmission electron microscopy, and nanoparticle tracking analysis were performed to characterize the EVs. The intracellular incorporation of EVs derived from PHK26 labeled and curcumin-primed or control hBMSC was tested by adding the labeled EVs to OA-CH cultures. OA-CH were pre-stimulated with IL-1ß, followed by Cur-EV and control EV treatment for 24 h and subsequent analysis of viability, apoptosis, and migration (scratch assay). Relative expression of selected anabolic and catabolic genes was assessed with qRT-PCR. Furthermore, WB was performed to evaluate phosphorylation of Erk1/2, PI3K/Akt, and p38MAPK in OA-CH. The effect of hsa-miR-126-3p expression on IL-1ß-induced OA-CH was determined using CTB-, Caspase 3/7-, live/dead assays, and WB. RESULTS: Cur-EVs promoted viability and reduced apoptosis of IL-1ß-stimulated OA-CH and attenuated IL-1ß-induced inhibition of migration. Furthermore, Cur-EVs increased gene expression of BCL2, ACAN, SOX9, and COL2A1 and decreased gene expression of IL1B, IL6, MMP13, and COL10A1 in IL-1ß-stimulated OA-CH. In addition, phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK, induced by IL-1ß, is prevented by Cur-EVs. Cur-EVs increased IL-1ß-reduced expression of hsa-miR-126-3p and hsa-miR-126-3p mimic reversed the effects of IL-1ß. CONCLUSION: Cur-EVs alleviated IL-1ß-induced catabolic effects on OA-CH by promoting viability and migration, reducing apoptosis and phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK thereby modulating pro-inflammatory signaling pathways. Treatment of OA-CH with Cur-EVs is followed by upregulation of expression of hsa-miR-126-3p which is involved in modulation of anabolic response of OA-CH. EVs may be considered as promising drug delivery vehicles of curcumin helping to alleviate OA.

A Large-Scale Bank of Organ Donor Bone Marrow and Matched Mesenchymal Stem Cells for Promoting Immunomodulation and Transplant Tolerance.

Induction of immune tolerance for solid organ and vascular composite allografts is the Holy Grail for transplantation medicine. This would obviate the need for life-long immunosuppression which is associated with serious adverse outcomes, such as infections, cancers, and renal failure. Currently the most promising means of tolerance induction is through establishing a mixed chimeric state by transplantation of donor hematopoietic stem cells; however, with the exception of living donor renal transplantation, the mixed chimerism approach has not achieved durable immune tolerance on a large scale in preclinical or clinical trials with other solid organs or vascular composite allotransplants (VCA). Ossium Health has established a bank of cryopreserved bone marrow (BM), termed "hematopoietic progenitor cell (HPC), Marrow," recovered from deceased organ donor vertebral bodies. This new source for hematopoietic cell transplant will be a valuable resource for treating hematological malignancies as well as for inducing transplant tolerance. In addition, we have discovered and developed a large source of mesenchymal stem (stromal) cells (MSC) tightly associated with the vertebral body bone fragment byproduct of the HPC, Marrow recovery process. Thus, these vertebral bone adherent MSC (vBA-MSC) are matched to the banked BM obtained from each donor, as opposed to third-party MSC, which enhances safety and potentially efficacy. Isolation and characterization of vBA-MSC from over 30 donors has demonstrated that the cells are no different than traditional BM-MSC; however, their abundance is >1,000-fold higher than obtainable from living donor BM aspirates. Based on our own unpublished data as well as reports published by others, MSC facilitate chimerism, especially at limiting hematopoietic stem and progenitor cell (HSPC) numbers and increase safety by controlling and/or preventing graft-vs.-host-disease (GvHD). Thus, vBA-MSC have the potential to facilitate mixed chimerism, promote complementary peripheral immunomodulatory functions and increase safety of BM infusions. Both HPC, Marrow and vBA-MSC have potential use in current VCA and solid organ transplant (SOT) tolerance clinical protocols that are amenable to "delayed tolerance." Current trials with HPC, Marrow are planned with subsequent phases to include vBA-MSC for tolerance of both VCA and SOT.

A quantitative serum biomarker of circulating collagen X effectively correlates with endochondral fracture healing.

Currently, there are no standardized methods for quantitatively measuring fracture repair. Physicians rely on subjective physical examinations and qualitative evaluation of radiographs to detect mineralized tissue. Since most fractures heal indirectly through a cartilage intermediate, these tools are limited in their diagnostic utility of early repair. Prior to converting to the bone, cartilage undergoes hypertrophic maturation, characterized by the deposition of a provisional collagen X matrix. The objective of this study was to characterize the kinetics of a novel collagen X biomarker relative to other biological measurements of fracture healing using a murine model of endochondral fracture repair in which a closed, mid-shaft tibia fracture was created using the classic drop-weight technique. Serum was collected 5 to 42 days post-fracture in male and female mice and compared to uninjured controls (n = 8-12). Collagen X in the serum was quantified using a recently validated ELISA-based bioassay ("Cxm")1 and compared to genetic and histological markers of fracture healing and inflammation. We found the Cxm biomarker reliably increased from baseline to a statistically unique peak 14 days post-fracture that then resolved to pre-fracture levels by 3 weeks following injury. The shape and timing of the Cxm curve followed the genetic and histological expression of collagen X but did not show a strong correlation with local inflammatory states. Assessment of fracture healing progress is crucial to making correct and timely clinical decisions for patients. This Cxm bioassay represents a minimally invasive, inexpensive technique that could provide reliable information on the biology of the fracture to significantly improve clinical care.

Norms for Clinical Use of CXM, a Real-Time Marker of Height Velocity.

CONTEXT: Height velocity (HV) is difficult to assess because growth is very slow. The current practice of calculating it from measurements taken at several-month intervals is insufficient for managing children with growth disorders. We identified a bone growth by-product (collagen X biomarker, CXM) in blood that in preliminary analysis in healthy children correlated strongly with conventionally determined HV and displayed a pattern resembling published norms for HV vs age. OBJECTIVE: The goal was to confirm our initial observations supporting the utility of CXM as an HV biomarker in a larger number of individuals and establish working reference ranges for future studies. DESIGN, SETTINGS, AND PARTICIPANTS: CXM was assessed in archived blood samples from 302 healthy children and 10 healthy adults yielding 961 CXM measurements. A total of 432 measurements were plotted by age, and sex-specific reference ranges were calculated. Serial values from 116 participants were plotted against observed HV. Matched plasma, serum, and dried blood spot readings were compared. RESULTS: A correlation of blood CXM with conventional HV was confirmed. Scatter plots of CXM vs age showed a similar pattern to current HV norms, and CXM levels demarcated the pubertal growth spurt both in girls and boys. CXM levels differed little in matched serum, plasma, and dried blood spot samples. CONCLUSIONS: Blood CXM offers a potential means to estimate HV in real time. Our results establish sex-specific, working reference ranges for assessing skeletal growth, especially over time. CXM stability in stored samples makes it well suited for retrospective studies.

Fibronectin Adherent Cell Populations Derived From Avascular and Vascular Regions of the Meniscus Have Enhanced Clonogenicity and Differentiation Potential Under Physioxia.

The meniscus is composed of an avascular inner region and vascular outer region. The vascular region has been shown to contain a progenitor population with multilineage differentiation capacity. Strategies facilitating the isolation and propagation of these progenitors can be used to develop cell-based meniscal therapies. Differential adhesion to fibronectin has been used to isolate progenitor populations from cartilage, while low oxygen or physioxia (2% oxygen) enhances the meniscal phenotype. This study aimed to isolate progenitor populations from the avascular and vascular meniscus using differential fibronectin adherence and examine their clonogenicity and differentiation potential under hyperoxia (20% oxygen) and physioxia (2% oxygen). Human vascular and avascular meniscus cells were seeded onto fibronectin-coated dishes for a short period and monitored for colony formation under either hyperoxia or physioxia. Non-fibronectin adherent meniscus cells were also expanded under both oxygen tension. Individual fibronectin adherent colonies were isolated and further expanded, until approximately ten population doublings (passage 3), whereby they underwent chondrogenic, osteogenic, and adipogenic differentiation. Physioxia enhances clonogenicity of vascular and avascular meniscus cells on plastic or fibronectin-coated plates. Combined differential fibronectin adhesion and physioxia isolated a progenitor population from both meniscus regions with trilineage differentiation potential compared to equivalent hyperoxia progenitors. Physioxia isolated progenitors had a significantly enhanced meniscus matrix content without the presence of collagen X. These results demonstrate that combined physioxia and fibronectin adherence can isolate and propagate a meniscus progenitor population that can potentially be used to treat meniscal tears or defects.

Exploring translational gaps between basic scientists, clinical researchers, clinicians, and consumers: Proceedings and recommendations arising from the 2020 mine the gap online workshop.

Objective: To provide a summary of the translational gaps in musculoskeletal research as identified in the Mine the Gap workshop and propose possible solutions. Methods: The Mine the Gap online workshop was hosted on October 14th and 15th, 2020. Five international panels, each comprised of a clinician, clinical researcher and basic scientist, presented gaps and proposed solutions for the themes of biomechanics, pain, biological measurements, phenotypes and imaging. This was followed by an interactive panel discussion with consumer insights. Results: A number of translational gaps and proposed solutions across each of the five themes were identified. A consumer panel provided constructive feedback highlighting the need for improved resources, communication and shared decision making, and treatment individualisation. Conclusion: This brief report provides a greater understanding of the diverse work and gaps relevant to fundamental/discovery scientists, clinical researchers and clinicians working across the musculoskeletal field. The numerous translational gaps highlight the need to improve communication and collaboration across the musculoskeletal field.