Janette N. Zara

NF-κB inhibits osteogenic differentiation of mesenchymal stem cells by promoting β-catenin degradation

Mesenchymal stem cell (MSC)-based transplantation is a promising therapeutic approach for bone regeneration and repair. In the realm of therapeutic bone regeneration, the defect or injured tissues are frequently inflamed with an abnormal expression of inflammatory mediators. Growing evidence suggests that proinflammatory cytokines inhibit osteogenic differentiation and bone formation. Thus, for successful MSC-mediated repair, it is important to overcome the inflammation-mediated inhibition of tissue regeneration. In this study, using genetic and chemical approaches, we found that proinflammatory cytokines TNF and IL-17 stimulated IκB kinase (IKK)–NF-κB and impaired osteogenic differentiation of MSCs. In contrast, the inhibition of IKK–NF-κB significantly enhanced MSC-mediated bone formation. Mechanistically, we found that IKK–NF-κB activation promoted β-catenin ubiquitination and degradation through induction of Smurf1 and Smurf2. To translate our basic findings to potential clinic applications, we showed that the IKK small molecule inhibitor, IKKVI, enhanced osteogenic differentiation of MSCs. More importantly, the delivery of IKKVI promoted MSC-mediated craniofacial bone regeneration and repair in vivo. Considering the well established role of NF-κB in inflammation and infection, our results suggest that targeting IKK–NF-κB may have dual benefits in enhancing bone regeneration and repair and inhibiting inflammation, and this concept may also have applicability in many other tissue regeneration situations.

Perivascular Stem Cells: A Prospectively Purified Mesenchymal Stem Cell Population for Bone Tissue Engineering

Adipose tissue is an ideal source of mesenchymal stem cells for bone tissue engineering: it is largely dispensable and readily accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which leads to unreliable bone formation. In the present study, we prospectively purified human perivascular stem cells (PSCs) from adipose tissue and compared their bone‐forming capacity with that of traditionally derived SVF. PSCs are a population (sorted by fluorescence‐activated cell sorting) of pericytes (CD146+CD34−CD45−) and adventitial cells (CD146−CD34+CD45−), each of which we have previously reported to have properties of mesenchymal stem cells. Here, we found that PSCs underwent osteogenic differentiation in vitro and formed bone after intramuscular implantation without the need for predifferentiation. We next sought to optimize PSCs for in vivo bone formation, adopting a demineralized bone matrix for osteoinduction and tricalcium phosphate particle formulation for protein release. Patient‐matched, purified PSCs formed significantly more bone in comparison with traditionally derived SVF by all parameters. Recombinant bone morphogenetic protein 2 increased in vivo bone formation but with a massive adipogenic response. In contrast, recombinant Nel‐like molecule 1 (NELL‐1; a novel osteoinductive growth factor) selectively enhanced bone formation. These studies suggest that adipose‐derived human PSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, PSCs are a stem cell‐based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy. Finally, NELL‐1 is a candidate growth factor able to induce human PSC osteogenesis.

The antimicrobial and osteoinductive properties of silver nanoparticle/poly (dl-lactic-co-glycolic acid)-coated stainless steel

Implant-associated bacterial infections are one of the most serious complications in orthopedic surgery. Treatment of these infections often requires multiple operations, device removal, long-term systemic antibiotics, and extended rehabilitation, and is frequently ineffective, leading to worse clinical outcomes and increased financial costs. In this study, we evaluated silver nanoparticle/poly(DL-lactic-co-glycolic acid) (PLGA)-coated stainless steel alloy(SNPSA) as a potential antimicrobial implant material. We found that SNPSA exhibited strong antibacterial activity in vitro and ex vivo, and promoted MC3T3-E1 pre-osteoblasts proliferation and maturation in vitro. Furthermore, SNPSA implants induced osteogenesis while suppressing bacterial survival in contaminated rat femoral canals. Our results indicate that SNPSA has simultaneous antimicrobial and osteoinductive properties that make it a promising therapeutic material in orthopedic surgery.

The use of BMP-2 coupled – Nanosilver-PLGA composite grafts to induce bone repair in grossly infected segmental defects

Healing of contaminated/infected bone defects is a significant clinical challenge. Prevalence of multi-antibiotic resistant organisms has renewed interest in the use of antiseptic silver as an effective, but less toxic antimicrobial with decreased potential for bacterial resistance. In this study, we demonstrated that metallic nanosilver particles (with a size of 20-40nm)-poly(lactic-co-glycolic acid) (PLGA) composite grafts have strong antibacterial properties. In addition, nanosilver particles-PLGA composite grafts did not inhibit adherence, proliferation, alkaline phosphatase activity, or mineralization of ongrowth MC3T3-E1 pre-osteoblasts compared to PLGA controls. Furthermore, nanosilver particles did not affect the osteoinductivity of bone morphogenetic protein 2 (BMP-2). Infected femoral defects implanted with BMP-2 coupled 2.0% nanosilver particles-PLGA composite grafts healed in 12 weeks without evidence of residual bacteria. In contrast, BMP-2 coupled PLGA control grafts failed to heal in the presence of continued bacterial colonies. Our results indicate that nanosilver of defined particle size is bactericidal without discernable in vitro and in vivo cytotoxicity or negative effects on BMP-2 osteoinductivity, making it an ideal antimicrobial for bone regeneration in infected wounds.

An Abundant Perivascular Source of Stem Cells for Bone Tissue Engineering

Adipose tissue is an ideal mesenchymal stem cell (MSC) source, as it is dispensable and accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which has disadvantages for tissue regeneration. In the present study, we prospectively purified human perivascular stem cells (PSCs) from n = 60 samples of human lipoaspirate and documented their frequency, viability, and variation with patient demographics. PSCs are a fluorescence‐activated cell sorting‐sorted population composed of pericytes (CD45−, CD146+, CD34−) and adventitial cells (CD45−, CD146−, CD34+), each of which we have previously reported to have properties of MSCs. Here, we found that PSCs make up, on average, 43.2% of SVF from human lipoaspirate (19.5% pericytes and 23.8% adventitial cells). These numbers were minimally changed by age, gender, or body mass index of the patient or by length of refrigerated storage time between liposuction and processing. In a previous publication, we observed that human PSCs (hPSCs) formed significantly more bone in vivo in comparison with unsorted human SVF (hSVF) in an intramuscular implantation model. We now extend this finding to a bone injury model, observing that purified hPSCs led to significantly greater healing of mouse critical‐size calvarial defects than hSVF (60.9% healing as opposed to 15.4% healing at 2 weeks postoperative by microcomputed tomography analysis). These studies suggest that adipose‐derived hPSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, hPSCs are a stem cell‐based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy.

The Role of NELL-1, a Growth Factor Associated With Craniosynostosis, in Promoting Bone Regeneration

Efforts to enhance bone regeneration in orthopedic and dental cases have grown steadily for the past decade, in line with increasingly sophisticated regenerative medicine. To meet the unprecedented demand for novel osteospecific growth factors with fewer adverse effects compared with those of existing adjuncts such as BMPs, our group has identified a craniosynostosis-associated secreted molecule, NELL-1, which is a potent growth factor that is highly specific to the osteochondral lineage, and has demonstrated robust induction of bone in multiple in vivo models from rodents to pre-clinical large animals. NELL-1 is preferentially expressed in osteoblasts under direct transcriptional control of Runx2, and is well-regulated during skeletal development. NELL-1/Nell-1 can promote orthotopic bone regeneration via either intramembranous or endochondral ossification, both within and outside of the craniofacial complex. Unlike BMP-2, Nell-1 cannot initiate ectopic bone formation in muscle, but can induce bone marrow stromal cells (BMSCs) to form bone in a mouse muscle pouch model, exhibiting specificity that BMPs lack. In addition, synergistic osteogenic effects of Nell-1 and BMP combotherapy have been observed, and are likely due to distinct differences in their signaling pathways. NELL-1’s unique role as a novel osteoinductive growth factor makes it an attractive alternative with promise for future clinical applications. [Note: NELL-1 and NELL-1 indicate the human gene and protein, respectively; Nell-1 and Nell-1 indicate the mouse gene and protein, respectively.]

Additive Effects of Sonic Hedgehog and Nell-1 Signaling in Osteogenic Versus Adipogenic Differentiation of Human Adipose-Derived Stromal Cells

A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the pro-osteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N+NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.

Nell-1 Enhances Bone Regeneration in a Rat Critical-Sized Femoral Segmental Defect Model

Background: Effective regeneration of bone is critical for fracture repair and incorporation and healing of bone grafts used during orthopedic, dental, and craniofacial reconstructions. Nel-like molecule-1 (Nell-1) is a secreted protein identified from prematurely fused cranial sutures of craniosynostosis patients that has been found to specifically stimulate osteogenic cell differentiation and bone formation. To test the in vivo osteoinductive capacity of Nell-1, a critical-sized femoral segmental defect model in athymic rats was used. Methods: A 6-mm defect, which predictably leads to nonunion if left untreated, was created in the left femur of each rat. Three treatment groups (n = 8 each) were created consisting of rats treated with (1) 1.5 mg/ml Nell-1, (2) 0.6 mg/ml Nell-1, and (3) phosphate-buffered saline only as a Nell-free control. Phosphate-buffered saline or Nell-1 was mixed with demineralized bone matrix as a carrier before implantation. All animals were euthanized 12 weeks after surgery, and bone regeneration was evaluated using radiographic, three-dimensional micro–computed tomographic, and histologic analysis. Results: Both Nell-1–treated groups had significantly greater bone formation compared with the Nell-free group, with bone volume increasing with increasing Nell-1 concentration. Conclusions: Nell-1 in a demineralized bone matrix carrier can significantly improve bone regeneration in a critical-sized femoral segmental defect in a dose-dependent manner. The results of this study demonstrate that Nell-1 is a potent osteospecific growth factor that warrants further investigation. Results also support the potential application of Nell-1 as a bone graft substitute in multiple clinical scenarios involving repair of critical bone loss when autograft bone is limited or unavailable.

Reprogramming of human fibroblasts into multipotent cells with a single ECM proteoglycan, fibromodulin

Pluripotent and/or multipotent stem cell-based therapeutics are a vital component of tissue engineering and regenerative medicine. The generation or isolation of safer and readily available stem cell sources will significantly aid clinical applications. We report here a technique using a single molecule, recombinant human fibromodulin protein (FMOD), to reprogram human fibroblasts into multipotent cells. Like virally-induced pluripotent stem (iPS) cells, FMOD reprogrammed (FReP) cells express pluripotency markers, form embryoid bodies (EBs), and differentiate into ectoderm, mesoderm, and endoderm derivatives in vitro. Notably, FReP cells regenerate muscle and bone tissues but do not generate teratomas in vivo. Unlike iPS cells, undifferentiated FReP cells proliferate slowly and express low proto-oncogene c-MYC and unexpectedly high levels of cyclin-dependent kinase inhibitors p15(Ink4B) and p21(WAF1/Cip1). Remarkably, in a fashion reminiscent of quiescent stem cells, the slow replicative phenotype of undifferentiated FReP cells reverses after differentiation induction, with differentiating FReP cells proliferating faster and expressing less p15(Ink4B) and p21(WAF1/Cip1) than differentiating iPS cells. Overall, single protein, FMOD-based, cell reprograming bypasses the risks of mutation, gene instability, and malignancy associated with genetically-modified iPS cells, and provides an alternative strategy for engineering patient-specific multipotent cells for basic research and therapeutic application.

Delayed wound closure in fibromodulin-deficient mice is associated with increased TGF-β3 signaling.

Fibromodulin (FMOD), a small leucine-rich proteoglycan, mediates scarless fetal skin wound repair through, in part, transforming growth factor-β (TGF-β) modulation. Using an adult fmod-null (fmod(-/-)) mouse model, this study further elucidates the interplay between FMOD and TGF-β expression during cutaneous repair and scar formation. Full-thickness skin wounds on fmod(-/-) and wild-type (WT) mice were closed primarily and analyzed. Histomorphometry revealed delayed dermal cell migration leading to delayed wound closure and significantly increased scar size in fmod(-/-) mice relative to WT, which was partially rescued by exogenous FMOD administration. In addition, fmod(-/-) wounds exhibited early elevation (within 24  hours post-wounding) of type I and type II TGF-β receptors as well as unexpectedly high fibroblast expression of TGF-β3, a molecule with reported antifibrotic and antimigratory effects. Consistent with elevated fibroblastic TGF-β3, fmod(-/-) fibroblasts were significantly less motile than WT fibroblasts. fmod(-/-) fibroblasts were also more susceptible to migration inhibition by TGF-β3, leading to profound delays in dermal cell migration. Increased scarring in fmod(-/-) mice indicates that TGF-β3s antimotility effects predominate over its antifibrotic effects when high TGF-β3 levels disrupt early fibroblastic wound ingress. These studies demonstrate that FMOD presence is critical for proper temporospatial coordination of wound healing events and normal TGF-β bioactivity.