Publications by authors named "Aaron W James"

155 Publications

Development of a Biomaterial Scaffold Integrated with Osteoinductive Oxysterol Liposomes to Enhance Hedgehog Signaling and Bone Repair.

Mol Pharm 2021 04 24;18(4):1677-1689. Epub 2021 Mar 24.

Department of Pathology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, United States.

Bone repair requires the tightly regulated control of multiple intrinsic and extrinsic cell types and signaling pathways. One of the positive regulatory signaling pathways in membranous and endochondral bone healing is the Hedgehog (Hh) signaling family. Here, a novel therapeutic liposomal delivery vector was developed by self-assembly of an Hh-activating cholesterol analog with an emulsifier, along with the addition of Smoothened agonist (SAG) as a drug cargo, for the enhancement of Hh signaling in bone regeneration. The drug-loaded nanoparticulate agonists of Hh signaling were immobilized onto trabecular bone-mimetic apatite-coated 3D scaffolds using bioinspired polydopamine adhesives to ensure favorable microenvironments for cell growth and local therapeutic delivery. Results showed that SAG-loaded liposomes induced a significant and dose-dependent increase in Hh-mediated osteogenic differentiation, as evidenced by analysis of bone marrow stromal cells, and calvarial bone healing, as evidenced using all radiographic parameters and histomorphometric analyses. Moreover, favorable outcomes were achieved in comparison to standards of care, including collagen sponge-delivered rBMP2 or allograft bone. In summary, this study demonstrates using a nanoparticle packaged Hh small molecule as a widely applicable bone graft substitute for robust bone repair.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c01136DOI Listing
April 2021

Assessing the Bone-Forming Potential of Pericytes.

Methods Mol Biol 2021 ;2235:127-137

Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.

Human pericytes are a perivascular cell population with mesenchymal stem cell properties, present in all vascularized tissues. Human pericytes have a distinct immunoprofile, which may be leveraged for purposes of cell purification. Adipose tissue is the most commonly used cell source for human pericyte derivation. Pericytes can be isolated by FACS (fluorescence-activated cell sorting), most commonly procured from liposuction aspirates. Pericytes have clonal multilineage differentiation potential, and their potential utility for bone regeneration has been described across multiple animal models. The following review will discuss in vivo methods for assessing the bone-forming potential of purified pericytes. Potential models include (1) mouse intramuscular implantation, (2) mouse calvarial defect implantation, and (3) rat spinal fusion models. In addition, the presented surgical protocols may be used for the in vivo analysis of other osteoprogenitor cell types.
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http://dx.doi.org/10.1007/978-1-0716-1056-5_9DOI Listing
March 2021

Divergent effects of distinct perivascular cell subsets for intra-articular cell therapy in posttraumatic osteoarthritis.

J Orthop Res 2021 Jan 29. Epub 2021 Jan 29.

Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USA.

Intra-articular injection of mesenchymal stem cells has shown benefit for the treatment of osteoarthritis (OA). However, mesenchymal stem/stromal cells at the origin of these clinical results are heterogenous cell populations with limited cellular characterization. Here, two transgenic reporter mice were used to examine the differential effects of two precisely defined perivascular cell populations (Pdgfrα  and Pdgfrβ  cells) from white adipose tissue for alleviation of OA. Perivascular mesenchymal cells were isolated from transgenic Pdgfrα-and Pdgfrβ-CreER reporter animals and delivered as a one-time intra-articular dose to C57BL/6J mice after destabilization of the medial meniscus (DMM). Both Pdgfrα  and Pdgfrβ  cell preparations improved metrics of cartilage degradation and reduced markers of chondrocyte hypertrophy. While some similarities in cell distribution were identified within the synovial and perivascular spaces, injected Pdgfrα  cells remained in the superficial layers of articular cartilage, while Pdgfrβ  cells were more widely dispersed. Pdgfrβ  cell therapy prevented subchondral sclerosis induced by DMM, while Pdgfrα  cell therapy had no effect. In summary, while both cell therapies showed beneficial effects in the DMM model, important differences in cell incorporation, persistence, and subchondral sclerosis were identified.
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http://dx.doi.org/10.1002/jor.24997DOI Listing
January 2021

Author Correction: NELL-1 in the treatment of osteoporotic bone loss.

Nat Commun 2021 Jan 13;12(1):453. Epub 2021 Jan 13.

Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA and Orthopaedic Hospital, University of California, Los Angeles, California, 90095, USA.

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http://dx.doi.org/10.1038/s41467-021-20933-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806913PMC
January 2021

Reply to the Letter to the Editor.

Int J Surg Pathol 2021 Apr 12;29(2):230. Epub 2021 Jan 12.

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http://dx.doi.org/10.1177/1066896920985731DOI Listing
April 2021

Systemic DKK1 neutralization enhances human adipose-derived stem cell mediated bone repair.

Stem Cells Transl Med 2021 Apr 30;10(4):610-622. Epub 2020 Dec 30.

Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USA.

Progenitor cells from adipose tissue are able to induce bone repair; however, inconsistent or unreliable efficacy has been reported across preclinical and clinical studies. Soluble inhibitory factors, such as the secreted Wnt signaling antagonists Dickkopf-1 (DKK1), are expressed to variable degrees in human adipose-derived stem cells (ASCs), and may represent a targetable "molecular brake" on ASC mediated bone repair. Here, anti-DKK1 neutralizing antibodies were observed to increase the osteogenic differentiation of human ASCs in vitro, accompanied by increased canonical Wnt signaling. Human ASCs were next engrafted into a femoral segmental bone defect in NOD-Scid mice, with animals subsequently treated with systemic anti-DKK1 or isotype control during the repair process. Human ASCs alone induced significant but modest bone repair. However, systemic anti-DKK1 induced an increase in human ASC engraftment and survival, an increase in vascular ingrowth, and ultimately improved bone repair outcomes. In summary, anti-DKK1 can be used as a method to augment cell-mediated bone regeneration, and could be particularly valuable in the contexts of impaired bone healing such as osteoporotic bone repair.
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http://dx.doi.org/10.1002/sctm.20-0293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7980212PMC
April 2021

Mesenchymal VEGFA induces aberrant differentiation in heterotopic ossification.

Bone Res 2019 Dec 10;7(1):36. Epub 2019 Dec 10.

Department of Surgery, University of Michigan, Ann Arbor, MI, 48109, USA.

Heterotopic ossification (HO) is a debilitating condition characterized by the pathologic formation of ectopic bone. HO occurs commonly following orthopedic surgeries, burns, and neurologic injuries. While surgical excision may provide palliation, the procedure is often burdened with significant intra-operative blood loss due to a more robust contribution of blood supply to the pathologic bone than to native bone. Based on these clinical observations, we set out to examine the role of vascular signaling in HO. Vascular endothelial growth factor A (VEGFA) has previously been shown to be a crucial pro-angiogenic and pro-osteogenic cue during normal bone development and homeostasis. Our findings, using a validated mouse model of HO, demonstrate that HO lesions are highly vascular, and that VEGFA is critical to ectopic bone formation, despite lacking a contribution of endothelial cells within the developing anlagen.
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http://dx.doi.org/10.1038/s41413-019-0075-6DOI Listing
December 2019

Administration of TGF-ß Inhibitor Mitigates Radiation-induced Fibrosis in a Mouse Model.

Clin Orthop Relat Res 2021 Mar;479(3):468-474

I. Gans, J. M. El Abiad, A. S. Levin, C. D. Morris, Department of Orthopaedic Surgery, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Background: Radiation-induced fibrosis is a long-term adverse effect of external beam radiation therapy for cancer treatment that can cause pain, loss of function, and decreased quality of life. Transforming growth factor beta (TGF-β) is believed to be critical to the development of radiation-induced fibrosis, and TGF-β inhibition decreases the development of fibrosis. However, no treatment exists to prevent radiation-induced fibrosis. Therefore, we aimed to mitigate the development of radiation-induced fibrosis in a mouse model by inhibiting TGF-β.

Question/purposes: Does TGF-β inhibition decrease the development of muscle fibrosis induced by external beam radiation in a mouse model?

Methods: Twenty-eight 12-week-old male C57BL/6 mice were assigned randomly to three groups: irradiated mice treated with TGF-βi, irradiated mice treated with placebo, and control mice that received neither irradiation nor treatment. The irradiated mice received one 50-Gy fraction of radiation to the right hindlimb before treatment initiation. Mice treated with TGF-c (n = 10) received daily intraperitoneal injections of a small-molecule inhibitor of TGF-β (1 mg/kg) in a dimethyl sulfoxide vehicle for 8 weeks (seven survived to histologic analysis). Mice treated with placebo (n = 10) received daily intraperitoneal injections of only a dimethyl sulfoxide vehicle for 8 weeks (10 survived to histologic analysis). Control mice (n = 8) received neither radiation nor TGF-β treatment. Control mice were euthanized at 3 months because they were not expected to exhibit any changes related to treatment. Mice in the two treatment groups were euthanized 9 months after radiation, and the quadriceps of each thigh was sampled. Masson's trichome stain was used to assess muscle fibrosis. Slides were viewed at 10 × magnification using bright-field microscopy, and in a blinded fashion, five representative images per mouse were used to quantify fibrosis. The mean ± SD fibrosis pixel densities in the TGF-βi and radiation-only groups were compared using Mann-Whitney U tests. The ratio of fibrosis to muscle was calculated using the mean fibrosis per slide in the TGF-βi group to standardize measurements. Alpha was set at 0.05.

Results: The mean (± SD) percentage of fibrosis per slide was greater in the radiation-only group (1.2% ± 0.42%) than in the TGF-βi group (0.14% ± 0.09%) (odds ratio 0.12 [95% CI 0.07 to 0.20]; p < 0.001). Among control mice, mean fibrosis was 0.05% ± 0.02% per slide. Mice in the radiation-only group had 9.1 times the density of fibrosis as did mice in the TGF-βi group.

Conclusion: Our study provides preliminary evidence that the fibrosis associated with radiation therapy to a quadriceps muscle can be reduced by treatment with a TGF-β inhibitor in a mouse model.

Clinical Relevance: If these observations are substantiated by further investigation into the role of TGF-β inhibition on the development of radiation-induced fibrosis in larger animal models and humans, our results may aid in the development of novel therapies to mitigate this complication of radiation treatment.
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http://dx.doi.org/10.1097/CORR.0000000000001286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899598PMC
March 2021

Lysosomal protein surface expression discriminates fat- from bone-forming human mesenchymal precursor cells.

Elife 2020 10 12;9. Epub 2020 Oct 12.

Departments of Pathology, Johns Hopkins University, Baltimore, United States.

Tissue resident mesenchymal stem/stromal cells (MSCs) occupy perivascular spaces. Profiling human adipose perivascular mesenchyme with antibody arrays identified 16 novel surface antigens, including endolysosomal protein CD107a. Surface CD107a expression segregates MSCs into functionally distinct subsets. In culture, CD107a cells demonstrate high colony formation, osteoprogenitor cell frequency, and osteogenic potential. Conversely, CD107a cells include almost exclusively adipocyte progenitor cells. Accordingly, human CD107a cells drove dramatic bone formation after intramuscular transplantation in mice, and induced spine fusion in rats, whereas CD107a cells did not. CD107a protein trafficking to the cell surface is associated with exocytosis during early adipogenic differentiation. RNA sequencing also suggested that CD107a cells are precursors of CD107a cells. These results document the molecular and functional diversity of perivascular regenerative cells, and show that relocation to cell surface of a lysosomal protein marks the transition from osteo- to adipogenic potential in native human MSCs, a population of substantial therapeutic interest.
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http://dx.doi.org/10.7554/eLife.58990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7550188PMC
October 2020

Human perivascular stem cells prevent bone graft resorption in osteoporotic contexts by inhibiting osteoclast formation.

Stem Cells Transl Med 2020 12 22;9(12):1617-1630. Epub 2020 Jul 22.

Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USA.

The vascular wall stores mesenchymal progenitor cells which are able to induce bone regeneration, via direct and paracrine mechanisms. Although much is known regarding perivascular cell regulation of osteoblasts, their regulation of osteoclasts, and by extension utility in states of high bone resorption, is not known. Here, human perivascular stem cells (PSCs) were used as a means to prevent autograft resorption in a gonadectomy-induced osteoporotic spine fusion model. Furthermore, the paracrine regulation by PSCs of osteoclast formation was evaluated, using coculture, conditioned medium, and purified extracellular vesicles. Results showed that PSCs when mixed with autograft bone induce an increase in osteoblast:osteoclast ratio, promote bone matrix formation, and prevent bone graft resorption. The confluence of these factors resulted in high rates of fusion in an ovariectomized rat lumbar spine fusion model. Application of PSCs was superior across metrics to either the use of unpurified, culture-defined adipose-derived stromal cells or autograft bone alone. Under coculture conditions, PSCs negatively regulated osteoclast formation and did so via secreted, nonvesicular paracrine factors. Total RNA sequencing identified secreted factors overexpressed by PSCs which may explain their negative regulation of graft resorption. In summary, PSCs reduce osteoclast formation and prevent bone graft resorption in high turnover states such as gonadectomy-induced osteoporosis.
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http://dx.doi.org/10.1002/sctm.20-0152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695633PMC
December 2020

Immobilization after injury alters extracellular matrix and stem cell fate.

J Clin Invest 2020 10;130(10):5444-5460

Section of Plastic Surgery, Department of Surgery.

Cells sense the extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction, which alters cell maintenance, proliferation, and differentiation. Here we use a mouse model of trauma-induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact mesenchymal progenitor cell (MPC) fate. After injury, single-cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional coactivator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, and signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, whereas in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.
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http://dx.doi.org/10.1172/JCI136142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524473PMC
October 2020

Perivascular Fibro-Adipogenic Progenitor Tracing during Post-Traumatic Osteoarthritis.

Am J Pathol 2020 09 10;190(9):1909-1920. Epub 2020 Jun 10.

Department of Pathology, Johns Hopkins University, Baltimore, Maryland; Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA and Orthopaedic Hospital, Los Angeles, California. Electronic address:

Perivascular mural cells surround capillaries and microvessels and have diverse regenerative or fibrotic functions after tissue injury. Subsynovial fibrosis is a well-known pathologic feature of osteoarthritis, yet transgenic animals for use in visualizing perivascular cell contribution to fibrosis during arthritic changes have not been developed. Here, inducible Pdgfra-CreER reporter mice were subjected to joint-destabilization surgery to induce arthritic changes, and cell lineage was traced over an 8-week period with a focus on the joint-associated fat pad. Results showed that, at baseline, inducible Pdgfra reporter activity highlighted adventitial and, to a lesser extent, pericytic cells within the infrapatellar fat pad. Joint-destabilization surgery was associated with marked fibrosis of the infrapatellar fat pad, accompanied by an expansion of perivascular Pdgfra-expressing cellular descendants, many of which adopted α-smooth muscle actin expression. Gene expression analysis of microdissected infrapatellar fat pad confirmed enrichment in membrane-bound green fluorescent protein/Pdgfra-expressing cells, along with a gene signature that corresponded with injury-associated fibro-adipogenic progenitors. Our results highlight dynamic changes in joint-associated perivascular fibro-adipogenic progenitors during osteoarthritis.
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http://dx.doi.org/10.1016/j.ajpath.2020.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456743PMC
September 2020

Comparison of skeletal and soft tissue pericytes identifies CXCR4 bone forming mural cells in human tissues.

Bone Res 2020 22;8:22. Epub 2020 May 22.

Departments of Pathology, Johns Hopkins University, Baltimore, 21205 MD USA.

Human osteogenic progenitors are not precisely defined, being primarily studied as heterogeneous multipotent cell populations and termed mesenchymal stem cells (MSCs). Notably, select human pericytes can develop into bone-forming osteoblasts. Here, we sought to define the differentiation potential of CD146 human pericytes from skeletal and soft tissue sources, with the underlying goal of defining cell surface markers that typify an osteoblastogenic pericyte. CD146CD31CD45 pericytes were derived by fluorescence-activated cell sorting from human periosteum, adipose, or dermal tissue. Periosteal CD146CD31CD45 cells retained canonical features of pericytes/MSC. Periosteal pericytes demonstrated a striking tendency to undergo osteoblastogenesis in vitro and skeletogenesis in vivo, while soft tissue pericytes did not readily. Transcriptome analysis revealed higher CXCR4 signaling among periosteal pericytes in comparison to their soft tissue counterparts, and CXCR4 chemical inhibition abrogated ectopic ossification by periosteal pericytes. Conversely, enrichment of CXCR4 pericytes or stromal cells identified an osteoblastic/non-adipocytic precursor cell. In sum, human skeletal and soft tissue pericytes differ in their basal abilities to form bone. Diversity exists in soft tissue pericytes, however, and CXCR4 pericytes represent an osteoblastogenic, non-adipocytic cell precursor. Indeed, enrichment for CXCR4-expressing stromal cells is a potential new tactic for skeletal tissue engineering.
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http://dx.doi.org/10.1038/s41413-020-0097-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244476PMC
May 2020

Endogenous CCN family member WISP1 inhibits trauma-induced heterotopic ossification.

JCI Insight 2020 07 9;5(13). Epub 2020 Jul 9.

Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USA.

Heterotopic ossification (HO) is defined as abnormal differentiation of local stromal cells of mesenchymal origin, resulting in pathologic cartilage and bone matrix deposition. Cyr61, CTGF, Nov (CCN) family members are matricellular proteins that have diverse regulatory functions on cell proliferation and differentiation, including the regulation of chondrogenesis. However, little is known regarding CCN family member expression or function in HO. Here, a combination of bulk and single-cell RNA sequencing defined the dynamic temporospatial pattern of CCN family member induction within a mouse model of trauma-induced HO. Among CCN family proteins, Wisp1 (also known as Ccn4) was most upregulated during the evolution of HO, and Wisp1 expression corresponded with chondrogenic gene profile. Immunohistochemistry confirmed WISP1 expression across traumatic and genetic HO mouse models as well as in human HO samples. Transgenic Wisp1LacZ/LacZ knockin animals showed an increase in endochondral ossification in HO after trauma. Finally, the transcriptome of Wisp1-null tenocytes revealed enrichment in signaling pathways, such as the STAT3 and PCP signaling pathways, that may explain increased HO in the context of Wisp1 deficiency. In sum, CCN family members, and in particular Wisp1, are spatiotemporally associated with and negatively regulate trauma-induced HO formation.
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http://dx.doi.org/10.1172/jci.insight.135432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406255PMC
July 2020

Clinicopathologic Analysis of Chondroblastoma in Adults: A Single-Institution Case Series.

Int J Surg Pathol 2021 Apr 2;29(2):120-128. Epub 2020 Jun 2.

1466Johns Hopkins University, Baltimore, MD, USA.

Chondroblastoma is a rare benign tumor of immature cartilage cells that generally occurs in an epiphyseal location of skeletally immature individuals. However, a few studies have reported cases in older patients. The purpose of this study was to evaluate the clinical, radiographic, and pathologic features of chondroblastoma in an adult population. The pathology archives of our institution were searched for cases of chondroblastoma diagnosed in patients ≥25 years of age. Of 14 patients identified, 8 were male and 6 were female with a median age of 34 years (range = 29-54 years). Most lesions occurred in short bones of hands and feet (N = 7, 50%), followed by the long tubular bones (N = 4, 28%). All demonstrated typical histologic features of chondroblastoma, but more extensive calcification, necrosis, and degenerative changes were also seen. At follow-up (median = 73.5 months), 2 patients (17%) had local recurrence. None had metastasis. In summary, chondroblastoma in adults tends to involve the short bones of the hands and feet and demonstrate histologic changes associated with long-standing growth of a benign tumor.
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http://dx.doi.org/10.1177/1066896920927794DOI Listing
April 2021

Anti-DKK1 Enhances the Early Osteogenic Differentiation of Human Adipose-Derived Stem/Stromal Cells.

Stem Cells Dev 2020 08 22;29(15):1007-1015. Epub 2020 Jun 22.

Department of Pathology and Johns Hopkins University, Baltimore, Maryland, USA.

Adipose-derived stem/stromal cells (ASCs) have been previously used for bone repair. However, significant cell heterogeneity exists within the ASC population, which has the potential to result in unreliable bone tissue formation and/or low efficacy. Although the use of cell sorting to lower cell heterogeneity is one method to improve bone formation, this is a technically sophisticated and costly process. In this study, we tried to find a simpler and more deployable solution-blocking antiosteogenic molecule Dickkopf-1 (DKK1) to improve osteogenic differentiation. Human adipose-derived stem cells were derived from = 5 samples of human lipoaspirate. In vitro, anti-DKK1 treatment, but not anti-sclerostin (SOST), promoted ASC osteogenic differentiation, assessed by alizarin red staining and real-time polymerase chain reaction (qPCR). Increased canonical Wnt signaling was confirmed after anti-DKK1 treatment. Expression levels of peaked during early osteogenic differentiation (day 3). Concordantly, anti-DKK1 supplemented early (day 3 or before), but not later (day 7) during osteogenic differentiation positively regulated osteoblast formation. Finally, anti-DKK1 led to increased transcript abundance of the Wnt inhibitor SOST, potentially representing a compensatory cellular mechanism. In sum, DKK1 represents a targetable "molecular brake" on the osteogenic differentiation of human ASC. Moreover, release of this brake by neutralizing anti-DKK1 antibody treatment at least partially rescues the poor bone-forming efficacy of ASC.
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http://dx.doi.org/10.1089/scd.2020.0070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7410293PMC
August 2020

A Neurotrophic Mechanism Directs Sensory Nerve Transit in Cranial Bone.

Cell Rep 2020 05;31(8):107696

Department of Pathology, Johns Hopkins University, Baltimore, MD 21205, USA. Electronic address:

The flat bones of the skull are densely innervated during development, but little is known regarding their role during repair. We describe a neurotrophic mechanism that directs sensory nerve transit in the mouse calvaria. Patent cranial suture mesenchyme represents an NGF (nerve growth factor)-rich domain, in which sensory nerves transit. Experimental calvarial injury upregulates Ngf in an IL-1β/TNF-α-rich defect niche, with consequent axonal ingrowth. In calvarial osteoblasts, IL-1β and TNF-α stimulate Ngf and downstream NF-κB signaling. Locoregional deletion of Ngf delays defect site re-innervation and blunted repair. Genetic disruption of Ngf among LysM-expressing macrophages phenocopies these observations, whereas conditional knockout of Ngf among Pdgfra-expressing cells does not. Finally, inhibition of TrkA catalytic activity similarly delays re-innervation and repair. These results demonstrate an essential role of NGF-TrkA signaling in bone healing and implicate macrophage-derived NGF-induced ingrowth of skeletal sensory nerves as an important mediator of this repair.
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http://dx.doi.org/10.1016/j.celrep.2020.107696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335423PMC
May 2020

Five Decades Later, Are Mesenchymal Stem Cells Still Relevant?

Front Bioeng Biotechnol 2020 28;8:148. Epub 2020 Feb 28.

MRC Centre for Regenerative Medicine and Centre for Cardiovascular Science, The University of Edinburgh, Edinburgh, United Kingdom.

Mesenchymal stem cells are culture-derived mesodermal progenitors isolatable from all vascularized tissues. In spite of multiple fundamental, pre-clinical and clinical studies, the native identity and role in tissue repair of MSCs have long remained elusive, with MSC selection from total cell suspensions essentially unchanged as a mere primary culture for half a century. Recent investigations have helped understand the tissue origin of these progenitor cells, and uncover alternative effects of MSCs on tissue healing growth factor secretion and interaction with the immune system. In this review, we describe current trends in MSC biology and discuss how these may improve the use of these therapeutic cells in tissue engineering and regenerative medicine.
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http://dx.doi.org/10.3389/fbioe.2020.00148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058632PMC
February 2020

Platelet-derived growth factor receptor-β (PDGFRβ) lineage tracing highlights perivascular cell to myofibroblast transdifferentiation during post-traumatic osteoarthritis.

J Orthop Res 2020 11 9;38(11):2484-2494. Epub 2020 Mar 9.

Department of Pathology, Johns Hopkins University, Baltimore, Maryland.

Pericytes ubiquitously surround capillaries and microvessels within vascularized tissues and have diverse functions after tissue injury. In addition to regulation of angiogenesis and tissue regeneration after injury, pericytes also contribute to organ fibrosis. Destabilization of the medial meniscus (DMM) phenocopies post-traumatic osteoarthritis, yet little is known regarding the impact of DMM surgery on knee joint-associated pericytes and their cellular descendants. Here, inducible platelet-derived growth factor receptor-β (PDGFRβ)-CreER reporter mice were subjected to DMM surgery, and lineage tracing studies performed over an 8-week period. Results showed that at baseline PDGFRβ reporter activity highlights abluminal perivascular cells within synovial and infrapatellar fat pad (IFP) tissues. DMM induces a temporospatially patterned increase in vascular density within synovial and subsynovial tissues. Marked vasculogenesis within IFP was accompanied by expansion of PDGFRβ reporter perivascular cell numbers, detachment of mGFP descendants from vessel walls, and aberrant adoption of myofibroblastic markers among mGFP cells including α-SMA, ED-A, and TGF-β1. At later timepoints, fibrotic changes and vascular maturation occurred within subsynovial tissues, with the redistribution of PDGFRβ cellular descendants back to their perivascular niche. In sum, PDGFRβ lineage tracing allows for tracing of perivascular cell fate within the diarthrodial joint. Further, destabilization of the joint induces vascular and fibrogenic changes of the IFP accompanied by perivascular to myofibroblast transdifferentiation.
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http://dx.doi.org/10.1002/jor.24648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7483913PMC
November 2020

Regulation of heterotopic ossification by monocytes in a mouse model of aberrant wound healing.

Nat Commun 2020 02 5;11(1):722. Epub 2020 Feb 5.

Section of Plastic Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI, 48109, USA.

Heterotopic ossification (HO) is an aberrant regenerative process with ectopic bone induction in response to musculoskeletal trauma, in which mesenchymal stem cells (MSC) differentiate into osteochondrogenic cells instead of myocytes or tenocytes. Despite frequent cases of hospitalized musculoskeletal trauma, the inflammatory responses and cell population dynamics that regulate subsequent wound healing and tissue regeneration are still unclear. Here we examine, using a mouse model of trauma-induced HO, the local microenvironment of the initial post-injury inflammatory response. Single cell transcriptome analyses identify distinct monocyte/macrophage populations at the injury site, with their dynamic changes over time elucidated using trajectory analyses. Mechanistically, transforming growth factor beta-1 (TGFβ1)-producing monocytes/macrophages are associated with HO and aberrant chondrogenic progenitor cell differentiation, while CD47-activating peptides that reduce systemic macrophage TGFβ levels and help ameliorate HO. Our data thus implicate CD47 activation as a therapeutic approach for modulating monocyte/macrophage phenotypes, MSC differentiation and HO formation during wound healing.
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http://dx.doi.org/10.1038/s41467-019-14172-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002453PMC
February 2020

Mesenchymal VEGFA induces aberrant differentiation in heterotopic ossification.

Bone Res 2019 10;7:36. Epub 2019 Dec 10.

1Department of Surgery, University of Michigan, Ann Arbor, MI 48109 USA.

Heterotopic ossification (HO) is a debilitating condition characterized by the pathologic formation of ectopic bone. HO occurs commonly following orthopedic surgeries, burns, and neurologic injuries. While surgical excision may provide palliation, the procedure is often burdened with significant intra-operative blood loss due to a more robust contribution of blood supply to the pathologic bone than to native bone. Based on these clinical observations, we set out to examine the role of vascular signaling in HO. Vascular endothelial growth factor A (VEGFA) has previously been shown to be a crucial pro-angiogenic and pro-osteogenic cue during normal bone development and homeostasis. Our findings, using a validated mouse model of HO, demonstrate that HO lesions are highly vascular, and that VEGFA is critical to ectopic bone formation, despite lacking a contribution of endothelial cells within the developing anlagen.
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http://dx.doi.org/10.1038/s41413-019-0075-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904752PMC
December 2019

PDGFRα marks distinct perivascular populations with different osteogenic potential within adipose tissue.

Stem Cells 2020 02 19;38(2):276-290. Epub 2019 Nov 19.

Department of Pathology, Johns Hopkins University, Baltimore, Maryland.

The perivascular niche within adipose tissue is known to house multipotent cells, including osteoblast precursors. However, the identity of perivascular subpopulations that may mineralize or ossify most readily is not known. Here, we utilize inducible PDGFRα (platelet-derived growth factor alpha) reporter animals to identify subpopulations of perivascular progenitor cells. Results showed that PDGFRα-expressing cells are present in four histologic niches within inguinal fat, including two perivascular locations. PDGFRα cells are most frequent within the tunica adventitia of arteries and veins, where PDGFRα cells populate the inner aspects of the adventitial layer. Although both PDGFRα and PDGFRα fractions are multipotent progenitor cells, adipose tissue-derived PDGFRα stromal cells proliferate faster and mineralize to a greater degree than their PDGFRα counterparts. Likewise, PDGFRα ectopic implants reconstitute the perivascular niche and ossify to a greater degree than PDGFRα cell fractions. Adventicytes can be further grouped into three distinct groups based on expression of PDGFRα and/or CD34. When further partitioned, adventicytes co-expressing PDGFRα and CD34 represented a cell fraction with the highest mineralization potential. Long-term tracing studies showed that PDGFRα-expressing adventicytes give rise to adipocytes, but not to other cells within the vessel wall under homeostatic conditions. However, upon bone morphogenetic protein 2 (BMP2)-induced ossicle formation, descendants of PDGFRα cells gave rise to osteoblasts, adipocytes, and "pericyte-like" cells within the ossicle. In sum, PDGFRα marks distinct perivascular osteoprogenitor cell subpopulations within adipose tissue. The identification of perivascular osteoprogenitors may contribute to our improved understanding of pathologic mineralization/ossification.
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http://dx.doi.org/10.1002/stem.3108DOI Listing
February 2020

CD10 expression identifies a subset of human perivascular progenitor cells with high proliferation and calcification potentials.

Stem Cells 2020 02 19;38(2):261-275. Epub 2019 Nov 19.

MRC Center for Regenerative Medicine and Center for Cardiovascular Science, University of Edinburgh, Scotland, UK.

The tunica adventitia ensheathes arteries and veins and contains presumptive mesenchymal stem cells (MSCs) involved in vascular remodeling. We show here that a subset of human adventitial cells express the CD10/CALLA cell surface metalloprotease. Both CD10 and CD10 adventitial cells displayed phenotypic features of MSCs when expanded in culture. However, CD10 adventitial cells exhibited higher proliferation, clonogenic and osteogenic potentials in comparison to their CD10 counterparts. CD10 adventitial cells increased expression of the cell cycle protein CCND2 via ERK1/2 signaling and osteoblastogenic gene expression via NF-κB signaling. CD10 expression was upregulated in adventitial cells through sonic hedgehog-mediated GLI1 signaling. These results suggest that CD10, which marks rapidly dividing cells in other normal and malignant cell lineages, plays a role in perivascular MSC function and cell fate specification. These findings also point to a role for CD10 perivascular cells in vascular remodeling and calcification.
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http://dx.doi.org/10.1002/stem.3112DOI Listing
February 2020

Fracture repair requires TrkA signaling by skeletal sensory nerves.

J Clin Invest 2019 12;129(12):5137-5150

Department of Pathology, Johns Hopkins University, Baltimore, Maryland, USA.

Bone is richly innervated by nerve growth factor-responsive (NGF-responsive) tropomyosin receptor kinase A-expressing (TrKa-expressing) sensory nerve fibers, which are required for osteochondral progenitor expansion during mammalian skeletal development. Aside from pain sensation, little is known regarding the role of sensory innervation in bone repair. Here, we characterized the reinnervation of tissue following experimental ulnar stress fracture and assessed the impact of loss of TrkA signaling in this process. Sequential histological data obtained in reporter mice subjected to fracture demonstrated a marked upregulation of NGF expression in periosteal stromal progenitors and fracture-associated macrophages. Sprouting and arborization of CGRP+TrkA+ sensory nerve fibers within the reactive periosteum in NGF-enriched cellular domains were evident at time points preceding periosteal vascularization, ossification, and mineralization. Temporal inhibition of TrkA catalytic activity by administration of 1NMPP1 to TrkAF592A mice significantly reduced the numbers of sensory fibers, blunted revascularization, and delayed ossification of the fracture callus. We observed similar deficiencies in nerve regrowth and fracture healing in a mouse model of peripheral neuropathy induced by paclitaxel treatment. Together, our studies demonstrate an essential role of TrkA signaling for stress fracture repair and implicate skeletal sensory nerves as an important upstream mediator of this repair process.
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http://dx.doi.org/10.1172/JCI128428DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6877307PMC
December 2019

Comparison of Human Tissue Microarray to Human Pericyte Transcriptome Yields Novel Perivascular Cell Markers.

Stem Cells Dev 2019 09 1;28(18):1214-1223. Epub 2019 Aug 1.

Department of Pathology, Johns Hopkins University, Baltimore, Maryland.

Human perivascular progenitor cells, including pericytes, are well-described multipotent mesenchymal cells giving rise to mesenchymal stem cells in culture. Despite the unique location of pericytes, specific antigens to distinguish human pericytes from other cell types are few. Here, we employed a human tissue microarray (Human Protein Atlas) to identify proteins that are strongly and specifically expressed in a pericytic location within human adipose tissue. Next, these results were cross-referenced with RNA sequencing data from human adipose tissue pericytes, as defined as a fluorescence activated cell sorting (FACS) purified CD146CD34CD31CD45 cell population. Results showed that from 105,532 core biopsies of soft tissue, 229 proteins showed strong and specific perivascular immunoreactivity, the majority of which (155) were present in the . Next, cross-referencing with the transcriptome of FACS-derived CD146 pericytes yielded 25 consistently expressed genes/proteins, including 18 novel antigens. A majority of these transcripts showed maintained expression after culture propagation (56% of genes). Interestingly, many novel antigens within pericytes are regulators of osteogenic differentiation. In sum, our study demonstrates the existence of novel pericyte markers, some of which are conserved in culture that may be useful for future efforts to typify, isolate, and characterize human pericytes.
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http://dx.doi.org/10.1089/scd.2019.0106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751392PMC
September 2019

Differential Vascularity in Genetic and Nonhereditary Heterotopic Ossification.

Int J Surg Pathol 2019 Dec 28;27(8):859-867. Epub 2019 Jun 28.

Johns Hopkins University, Baltimore, MD, USA.

Nonhereditary heterotopic ossification (NHO) is a common complication of trauma. Progressive osseous heteroplasia (POH) and fibrodysplasia ossificans progressiva (FOP) are rare genetic causes of heterotopic bone. In this article, we detail the vascular patterning associated with genetic versus NHO. Vascular histomorphometric analysis was performed on patient samples from POH, FOP, and NHO. Endpoints for analysis included blood vessel (BV) number, area, density, size, and wall thickness. Results demonstrated conserved temporal dynamic changes in vascularity across all heterotopic ossification lesions. Immature areas had the highest BV number, while the more mature foci had the highest BV area. Most vascular parameters were significantly increased in genetic as compared with NHO. In sum, both genetic and NHO show temporospatial variation in vascularity. These findings suggest that angiogenic pathways are potential therapeutic targets in both genetic and nonhereditary forms of heterotopic ossification.
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http://dx.doi.org/10.1177/1066896919857135DOI Listing
December 2019

Pericytes in Sarcomas and Other Mesenchymal Tumors.

Adv Exp Med Biol 2019 ;1147:109-124

Division of Surgical Pathology, Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.

Tumors of mesenchymal origin are a diverse group, with >130 distinct entities currently recognized by the World Health Organization. A subset of mesenchymal tumors grow or invade in a perivascular fashion, and their potential relationship to pericytes is a matter of ongoing interest. In fact, multiple intersections exist between pericytes and tumors of mesenchymal origin. First, pericytes are the likely cell of origin for a group of mesenchymal tumors with a common perivascular growth pattern. These primarily benign tumors grow in a perivascular fashion and diffusely express canonical pericyte markers such as CD146, smooth muscle actin (SMA), platelet-derived growth factor receptor beta (PDGFR-β), and RGS5. These benign tumors include glomus tumor, myopericytoma, angioleiomyoma, and myofibroma. Second and as suggested by animal models, pericytes may give rise to malignant sarcomas. This is not a suggestion that all sarcomas within a certain subtype arise from pericytes, but that genetic modifications within a pericyte cell type may give rise to sarcomas. Third, mesenchymal tumors that are likely not a pericyte derivative co-opt pericyte markers in certain contexts. These include the PEComa family of tumors and liposarcoma. Fourth and finally, as "guardians" that enwrap the microvasculature, nonneoplastic pericytes may be important in sarcoma disease progression.
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http://dx.doi.org/10.1007/978-3-030-16908-4_4DOI Listing
August 2019

Heterotopic Ossification: A Comprehensive Review.

JBMR Plus 2019 Apr 27;3(4):e10172. Epub 2019 Feb 27.

Department of Pathology Johns Hopkins University Baltimore MD USA.

Heterotopic ossification (HO) is a diverse pathologic process, defined as the formation of extraskeletal bone in muscle and soft tissues. HO can be conceptualized as a tissue repair process gone awry and is a common complication of trauma and surgery. This comprehensive review seeks to synthesize the clinical, pathoetiologic, and basic biologic features of HO, including nongenetic and genetic forms. First, the clinical features, radiographic appearance, histopathologic diagnosis, and current methods of treatment are discussed. Next, current concepts regarding the mechanistic bases for HO are discussed, including the putative cell types responsible for HO formation, the inflammatory milieu and other prerequisite "niche" factors for HO initiation and propagation, and currently available animal models for the study of HO of this common and potentially devastating condition. © 2019 The Authors. published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbm4.10172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478587PMC
April 2019

Skeletogenic Capacity of Human Perivascular Stem Cells Obtained Via Magnetic-Activated Cell Sorting.

Tissue Eng Part A 2019 12 16;25(23-24):1658-1666. Epub 2019 Aug 16.

Department of Pathology, Johns Hopkins University, Baltimore, Maryland.

Human perivascular stem/stromal cells (PSC) are a multipotent mesenchymal progenitor cell population defined by their perivascular residence. PSC are increasingly studied for their application in skeletal regenerative medicine. PSC from subcutaneous white adipose tissue are most commonly isolated via fluorescence-activated cell sorting (FACS), and defined as a bipartite population of CD146CD34CD31CD45 pericytes and CD34CD146CD31CD45 adventitial cells. FACS poses several challenges for clinical translation, including requirements for facilities, equipment, and personnel. The purpose of this study is to identify if magnetic-activated cell sorting (MACS) is a feasible method to derive PSC, and to determine if MACS-derived PSC are comparable to our previous experience with FACS-derived PSC. In brief, CD146 pericytes and CD34 adventitial cells were enriched from human lipoaspirate using a multistep column approach. Next, cell identity and purity were analyzed by flow cytometry. multilineage differentiation studies were performed with MACS-defined PSC subsets. Finally, application was performed in nonhealing calvarial bone defects in mice. Results showed that human CD146 pericytes and CD34 adventitial cells may be enriched by MACS, with defined purity, anticipated cell surface marker expression, and capacity for multilineage differentiation. , MACS-derived PSC induce ossification of bone defects. These data document the feasibility of a MACS approach for the enrichment and application of PSC in the field of tissue engineering and regenerative medicine. Impact Statement Our findings suggest that perivascular stem/stromal cells, and in particular adventitial cells, may be isolated by magnetic-activated cell sorting and applied as an uncultured autologous stem cell therapy in a same-day setting for bone defect repair.
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http://dx.doi.org/10.1089/ten.TEA.2019.0031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6919244PMC
December 2019

Overlapping features of rapidly progressive osteoarthrosis and Charcot arthropathy.

J Orthop 2019 May-Jun;16(3):260-264. Epub 2019 Mar 12.

Department of Pathology, Johns Hopkins University, Ross Research Building, Room 524A, 720 Rutland Avenue, Baltimore, MD, 21205, United States.

Nerve growth factor (NGF) is the primary neurotrophin in the skeleton and a central mediator of skeletal pain. Recent trials of anti-NGF neutralizing antibodies have resulted in infrequent but well-described incidence of rapidly progressive osteoarthrosis (RPOA). Neuropathy, whether from syphilis or diabetes, is also associated with severe joint destruction, known as neuroarthropathy or Charcot joint. These commonalities of severe joint destruction with either loss of a neurotrophin (anti-NGF) or a deficit of functional skeletal innervation led us to examine our institutional case files for potential radio-pathologic overlap between RPOA and Charcot joint.
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http://dx.doi.org/10.1016/j.jor.2019.02.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6426701PMC
March 2019