Interaction between the nervous and skeletal systems.

The skeleton is one of the largest organ systems in the body and is richly innervated by the network of nerves. Peripheral nerves in the skeleton include sensory and sympathetic nerves. Crosstalk between bones and nerves is a hot topic of current research, yet it is not well understood. In this review, we will explore the role of nerves in bone repair and remodeling, as well as summarize the molecular mechanisms by which neurotransmitters regulate osteogenic differentiation. Furthermore, we discuss the skeleton's role as an endocrine organ that regulates the innervation and function of nerves by secreting bone-derived factors. An understanding of the interactions between nerves and bone can help to prevent and treat bone diseases caused by abnormal innervation or nerve function, develop new strategies for clinical bone regeneration, and improve patient outcomes.

NGF-p75 signaling coordinates skeletal cell migration during bone repair.

Bone regeneration following injury is initiated by inflammatory signals and occurs in association with infiltration by sensory nerve fibers. Together, these events are believed to coordinate angiogenesis and tissue reprogramming, but the mechanism of coupling immune signals to reinnervation and osteogenesis is unknown. Here, we found that nerve growth factor (NGF) is expressed following cranial bone injury and signals via p75 in resident mesenchymal osteogenic precursors to affect their migration into the damaged tissue. Mice lacking Ngf in myeloid cells demonstrated reduced migration of osteogenic precursors to the injury site with consequently delayed bone healing. These features were phenocopied by mice lacking p75 in Pdgfra+ osteoblast precursors. Single-cell transcriptomics identified mesenchymal subpopulations with potential roles in cell migration and immune response, altered in the context of p75 deletion. Together, these results identify the role of p75 signaling pathway in coordinating skeletal cell migration during early bone repair.

NGF-TrkA signaling dictates neural ingrowth and aberrant osteochondral differentiation after soft tissue trauma.

Pain is a central feature of soft tissue trauma, which under certain contexts, results in aberrant osteochondral differentiation of tissue-specific stem cells. Here, the role of sensory nerve fibers in this abnormal cell fate decision is investigated using a severe extremity injury model in mice. Soft tissue trauma results in NGF (Nerve growth factor) expression, particularly within perivascular cell types. Consequently, NGF-responsive axonal invasion occurs which precedes osteocartilaginous differentiation. Surgical denervation impedes axonal ingrowth, with significant delays in cartilage and bone formation. Likewise, either deletion of Ngf or two complementary methods to inhibit its receptor TrkA (Tropomyosin receptor kinase A) lead to similar delays in axonal invasion and osteochondral differentiation. Mechanistically, single-cell sequencing suggests a shift from TGFß to FGF signaling activation among pre-chondrogenic cells after denervation. Finally, analysis of human pathologic specimens and databases confirms the relevance of NGF-TrkA signaling in human disease. In sum, NGF-mediated TrkA-expressing axonal ingrowth drives abnormal osteochondral differentiation after soft tissue trauma. NGF-TrkA signaling inhibition may have dual therapeutic use in soft tissue trauma, both as an analgesic and negative regulator of aberrant stem cell differentiation.

Assessing the Bone-Forming Potential of Pericytes.

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.

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

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.

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

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, CD107alow cells demonstrate high colony formation, osteoprogenitor cell frequency, and osteogenic potential. Conversely, CD107ahigh cells include almost exclusively adipocyte progenitor cells. Accordingly, human CD107alow cells drove dramatic bone formation after intramuscular transplantation in mice, and induced spine fusion in rats, whereas CD107ahigh cells did not. CD107a protein trafficking to the cell surface is associated with exocytosis during early adipogenic differentiation. RNA sequencing also suggested that CD107alow cells are precursors of CD107ahigh 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.

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

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.

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

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. CD146+CD31-CD45- pericytes were derived by fluorescence-activated cell sorting from human periosteum, adipose, or dermal tissue. Periosteal CD146+CD31-CD45- 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.

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

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.

A Neurotrophic Mechanism Directs Sensory Nerve Transit in Cranial Bone.

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-1ß/TNF-α-rich defect niche, with consequent axonal ingrowth. In calvarial osteoblasts, IL-1ß 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.

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

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-ß (PDGFRß)-CreERT2 reporter mice were subjected to DMM surgery, and lineage tracing studies performed over an 8-week period. Results showed that at baseline PDGFRß 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 PDGFRß 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-ß1. At later timepoints, fibrotic changes and vascular maturation occurred within subsynovial tissues, with the redistribution of PDGFRß+ cellular descendants back to their perivascular niche. In sum, PDGFRß 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.

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

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.

Fracture repair requires TrkA signaling by skeletal sensory nerves.

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.

Pericytes in Sarcomas and Other Mesenchymal Tumors.

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-ß), 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.

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

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 CD146+CD34-CD31-CD45- pericytes and CD34+CD146-CD31-CD45- 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. In vitro multilineage differentiation studies were performed with MACS-defined PSC subsets. Finally, in vivo application was performed in nonhealing calvarial bone defects in Scid 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. In vivo, 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.

Overlapping features of rapidly progressive osteoarthrosis and Charcot arthropathy.

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.

Age dependent effects of NELL-1 isoforms on bone marrow stromal cells.

NELL-1 is an osteogenic protein first discovered to control ossification of the cranium. NELL-1 exists in at least two isoforms. The full-length NELL-1 contains 810 amino acid (aa) (NELL-1810), the N-terminal-truncated NELL-1 isoform contains 570 aa (NELL-1570). The differences in cellular effects between NELL-1 isoforms are not well understood. Methods: Here, BMSC were derived from adult or aged mice, followed by overexpression of NELL-1810 or NELL-1570. Cell morphology, proliferation, and gene expression were examined. Results/Conclusions: Overall, the proliferative effect of NELL-1570 was age dependent, showing prominent induction in adult but not aged mice.

Relative contributions of adipose-resident CD146+ pericytes and CD34+ adventitial progenitor cells in bone tissue engineering.

Pericytes and other perivascular stem/stromal cells are of growing interest in the field of tissue engineering. A portion of perivascular cells are well recognized to have MSC (mesenchymal stem cell) characteristics, including multipotentiality, self-renewal, immunoregulatory functions, and diverse roles in tissue repair. Here, we investigate the differential but overlapping roles of two perivascular cell subsets in paracrine induction of bone repair. CD146+CD34-CD31-CD45-pericytes and CD34+CD146-CD31-CD45-adventitial cells were derived from human adipose tissue and applied alone or in combination to calvarial bone defects in mice. In vitro, osteogenic differentiation and tubulogenesis assays were performed using either fluorescence activated cell sorting-derived CD146+ pericytes or CD34+ adventitial cells. Results showed that CD146+ pericytes induced increased cord formation in vitro and angiogenesis in vivo in comparison with patient-matched CD34+ adventitial cells. In contrast, CD34+ adventitial cells demonstrated heightened paracrine-induced osteogenesis in vitro. When applied in a critical-size calvarial defect model in NOD/SCID mice, the combination treatment of CD146+ pericytes with CD34+ adventitial cells led to greater re-ossification than either cell type alone. In summary, adipose-derived CD146+ pericytes and CD34+ adventitial cells display functionally distinct yet overlapping and complementary roles in bone defect repair. Consequently, CD146+ pericytes and CD34+ adventitial cells may demonstrate synergistic bone healing when applied as a combination cellular therapy.

Pericytes for Therapeutic Bone Repair.

Besides seminal functions in angiogenesis and blood pressure regulation, microvascular pericytes possess a latent tissue regenerative potential that can be revealed in culture following transition into mesenchymal stem cells. Endowed with robust osteogenic potential, pericytes and other related perivascular cells extracted from adipose tissue represent a potent and abundant cell source for refined bone tissue engineering and improved cell therapies of fractures and other bone defects. The use of diverse bone formation assays in vivo, which include mouse muscle pocket osteogenesis and calvaria replenishment, rat and dog spine fusion, and rat non-union fracture healing, has confirmed the superiority of purified perivascular cells for skeletal (re)generation. As a surprising observation though, despite strong endogenous bone-forming potential, perivascular cells drive bone regeneration essentially indirectly, via recruitment by secreted factors of local osteo-progenitors.

WISP-1 drives bone formation at the expense of fat formation in human perivascular stem cells.

The vascular wall within adipose tissue is a source of mesenchymal progenitors, referred to as perivascular stem/stromal cells (PSC). PSC are isolated via fluorescence activated cell sorting (FACS), and defined as a bipartite population of pericytes and adventitial progenitor cells (APCs). Those factors that promote the differentiation of PSC into bone or fat cell types are not well understood. Here, we observed high expression of WISP-1 among human PSC in vivo, after purification, and upon transplantation in a bone defect. Next, modulation of WISP-1 expression was performed, using WISP-1 overexpression, WISP-1 protein, or WISP-1 siRNA. Results demonstrated that WISP-1 is expressed in the perivascular niche, and high expression is maintained after purification of PSC, and upon transplantation in a bone microenvironment. In vitro studies demonstrate that WISP-1 has pro-osteogenic/anti-adipocytic effects in human PSC, and that regulation of BMP signaling activity may underlie these effects. In summary, our results demonstrate the importance of the matricellular protein WISP-1 in regulation of the differentiation of human stem cell types within the perivascular niche. WISP-1 signaling upregulation may be of future benefit in cell therapy mediated bone tissue engineering, for the healing of bone defects or other orthopaedic applications.