Kang Ting

CSU Mentor, Long Beach, California, United States

Are you Kang Ting?

Claim your profile

Publications (112)352.22 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Neural epidermal growth factor-like (NEL)-like protein 1 (NELL-1) has been identified as an osteoinductive differentiation factor that promotes mesenchymal stem cell (MSC) osteogenic differentiation. In addition to full-length NELL-1, there are several NELL-1-related transcripts reported. We used rapid amplification of cDNA ends (RACE) to recover potential cDNA of NELL-1 isoforms. A NELL-1 isoform with the N-terminal 240 amino acid (aa) residues truncated was identified. While full-length NELL-1 that contains 810 aa residues (NELL-1810) plays an important role in embryologic skeletal development, the N-terminal-truncated NELL-1 isoform (NELL-1570) was expressed post-natally. Similar to NELL-1810, NELL-1570 induced MSC osteogenic differentiation. In addition, NELL-1570 significantly stimulated MSC proliferation in multiple MSC-like populations such as murine C3H10T1/2 MSC cell line, mouse primary MSCs, and perivascular stem cells (PSCs), which is a type of stem cells proposed as the perivascular origin of MSCs. In contrast, NELL-1810 demonstrated only limited stimulation of MSC proliferation. Similar to NELL-1810, NELL-1570 was found to be secreted from host cells. Both NELL-1570 expression lentiviral vector and column-purified recombinant protein NELL-1570 demonstrated almost identical effects in MSC proliferation and osteogenic differentiation, suggesting that NELL-1570 may function as a pro-osteogenic growth factor. In vivo, NELL-1570 induced significant calvarial defect regeneration accompanied by increased cell proliferation. Thus, NELL-1570 has the potential to be used for cell-based or hormone-based therapy of bone regeneration. Stem Cells 2014
    Stem Cells 11/2014; · 7.70 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Adipose tissue is an attractive source of mesenchymal stem cells (MSCs) because of its abundance and accessibility. We have previously defined a population of native MSCs termed perivascular stem cells (PSCs), purified from diverse human tissues, including adipose tissue. Human PSCs (hPSCs) are a bipartite cell population composed of pericytes (CD146+CD34-CD45-) and adventitial cells (CD146-CD34+CD45-), isolated by fluorescence-activated cell sorting and with properties identical to those of culture identified MSCs. Our previous studies showed that hPSCs exhibit improved bone formation compared with a sample-matched unpurified population (termed stromal vascular fraction); however, it is not known whether hPSCs would be efficacious in a spinal fusion model. To investigate, we evaluated the osteogenic potential of freshly sorted hPSCs without culture expansion and differentiation in a rat model of posterolateral lumbar spinal fusion. We compared increasing dosages of implanted hPSCs to assess for dose-dependent efficacy. All hPSC treatment groups induced successful spinal fusion, assessed by manual palpation and microcomputed tomography. Computerized biomechanical simulation (finite element analysis) further demonstrated bone fusion with hPSC treatment. Histological analyses showed robust endochondral ossification in hPSC-treated samples. Finally, we confirmed that implanted hPSCs indeed differentiated into osteoblasts and osteocytes; however, the majority of the new bone formation was of host origin. These results suggest that implanted hPSCs positively regulate bone formation via direct and paracrine mechanisms. In summary, hPSCs are a readily available MSC population that effectively forms bone without requirements for culture or predifferentiation. Thus, hPSC-based products show promise for future efforts in clinical bone regeneration and repair.
    Stem cells translational medicine. 08/2014;
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Osteoporosis is the most common metabolic disease of bone, resulting in significant worldwide morbidity. Currently, there are insufficient imaging modalities available to evaluate osteoporotic bones in small animal models. Here, we demonstrate the feasibility of using high resolution X-ray imaging as a comparable measure of bone degeneration to dual-energy X-ray absorptiometry (DXA) in an osteoporosis rodent model. At week 0, animals underwent either an ovariectomy (OVX) or sham procedure (SHAM). DXA analysis was performed weekly to confirm and compare the bone degenerative changes induced by OVX. A comparison using high resolution X-ray imaging (Faxitron(®)) was then performed postmortem due to need of soft tissue removal. Two regions of interest (ROIs) were utilized: the distal third of the femur and the lumbar spine (L4/L5). It was observed that SHAM animals maintained a relatively constant bone mineral density (BMD), in comparison to OVX animals, whereby a significant decrease in BMD was appreciated. Post mortem X-ray scans were performed and converted to 8-bit color and quantified. A high level of agreement with DXA quantifications was observed with X-ray quantifications, and a significant correlation between the radiopacity, visualized by color distributions, and the DXA BMD values between animal groups was evident. Our study demonstrates the applicability of high resolution X-ray imaging both qualitatively and quantitatively as a reliable approach for quantifying osteoporosis in rodent osteoporotic models. With DXA being a highly user dependent modality, our technique is a unique secondary methodology to verify DXA findings and minimize inter-observer variability.
    BioResearch open access. 08/2014; 3(4):192-6.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objectives: NELL-1 is a potent cytokine with pro-osteogenic and anti-osteoclastic effects. Specifically, it activates Wnt/β-catenin signaling and has been shown in multiple animal models to induce robust bone formation locally and systemically. Our previous systemic administration study using unmodified NELL-1 has shown successful increase of bone quality/quantity in both healthy and osteoporotic mice. However, an impractical q2d injection frequency limits NELL-1’s translation into a clinical application. For this reason, this study investigates the potential of PEGylation in improving NELL-1’s pharmacokinetics, while maintaining its bioactivity and osteogenic potential. Methods: First, we evaluated three PEGylation patterns(5K-linear, 20K-linear, and 40K-branched) for 1]NELL-1 thermostability via differential scanning fluorimetry(DSF), 2]half-life by using FITC conjugated NELL-1, and 3]in vitro osteogenic potential via mineralization studies in preosteoblast cell lines. Next, we investigated the capacity of systemically administered PEG-NELL-1(1.25mg/kg, q4d) to promote bone formation in vivo, in 3-months-old B6 mice(n=10). To monitor bone mineral density(BMD), dual-energy X-ray absorptiometry(DXA) was performed weekly, with regions-of-interest(ROI) being distal femurs. Animals were sacrificed at 4 weeks post-treatment, and were analyzed by micro-Computed Tomography(microCT), histology, and immunohistochemistry. Results: PEGylation of NELL-1 was observed to enhance protein thermostability from 49.75oC up to 63.42oC(27% increase) and increase half-life from 5.5 hours to 16.1-31.3 hours(up to 6 fold), while retaining in vitro protein bioactivity levels comparable to unmodified NELL-1. In addition, systemic PEG-NELL-1 administration in vivo showed DXA result of a gradual and significant, 17% increase in femoral BMD compared to pre-treatment values. Post-mortem microCT also showed significant increases in trabecular and cortical bone BMD and volumetric measurements in the femurs. Conclusion: PEGylation of NELL-1 significantly increases protein thermostability and half-life, while retaining NELL-1’s osteogenic potential in vitro and in vivo. These exciting findings present PEGylated NELL-1 as a novel platform technology for systemic treatment of various osteodeficient disorders including osteoporosis and dysostosis syndromes.
    IADR General Session and Exhibition 2014; 06/2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: Introduction: Skeletal aging is associated not only with alterations in osteoblast (OB) and osteoclast (OC) number and activity within the basic metabolic unit, but also with increased marrow adiposity. Peroxisome proliferator-activated receptor gamma (PPARγ) is commonly considered the master transcriptional regulator of adipogenesis, however it has known roles in osteoblast and osteoclast function as well. Here, we designed a lentiviral delivery system for PPARγ shRNA, and examined its effects in vitro on bone marrow stromal cells (BMSC) and in a mouse intramedullary injection model. Methods: PPARγ shRNA was delivered by a replication deficient lentiviral vector, after in vitro testing to confirm purity, concentration, and efficacy for Pparg transcript reduction. Next, control (GFP) lentivirus or PPARγ shRNA expressing lentivirus were delivered by transpatellar intramedullary injection into the femoral bone marrow of male SCID mice. Analyses included daily monitoring of animal health, and post mortem analysis at 4 weeks. Post mortem analyses included high resolution microcomputed tomography (microCT) reconstructions and analysis, routine histology and histomorphometric analysis, qRT-PCR analysis of Pparg transcript levels, and immunohistochemical analysis for markers of adipocytes (PPARγ, FABP4), osteoblasts (ALP, OCN), and osteoclasts (TRAP, Cathepsin K). Results: In vitro, PPARγ shRNA delivery significantly reduced Pparg expression in mouse BMSC, accompanied by a significant reduction in lipid droplet accumulation. In vivo, a near total reduction in mature marrow adipocytes was observed at four weeks post-injection. This was accompanied by significant reductions in adipocyte-specific markers. Parameters of trabecular bone were significantly increased by both microCT and histomorphometric analysis. By immunohistochemical staining and semi-quantification, a significant increase in OCN+ osteoblasts and decrease in TRAP+ multinucleated osteoclasts was observed with PPARγ shRNA treatment. Discussion: These findings suggest that acute loss of PPARγ in the bone marrow compartment has a significant role beyond anti-adipose effects. Specifically, we found pro-osteoblastogenic, anti-osteoclastic effects after PPARγ shRNA treatment, resulting in improved trabecular bone architecture. Future studies will examine the isolated and direct effects of PPARγ shRNA on OB and OC cell types, and may help determine if PPARγ antagonists are potential therapeutic agents for osteoporotic bone loss.
    Tissue Engineering Part A 04/2014; · 4.64 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objectives: Mesenchymal stem cell commitment to an osteoprogenitor lineage requires the activity of Runx2, a molecule implicated in the etiopathology of multiple congenital craniofacial anomalies. Our previous studies have shown that NELL-1 functions downstream of Runx2 in osteogenesis, which can partially rescues craniofacial defects in Runx2 haploinsufficient(Runx2(+/-)) mice. In this study we will specifically focus on the Runx2-Nell-1 functional interaction during chondrogenesis. Methods: Runx2(-/-)/CMV-Nell-1 mice were generated and analyzed. Immunohistochemical staining of newborn limbs were performed and compared to Runx2(-/-) and wildtype(WT) mice. For ex-vivo, the limb buds were isolated from Runx2(-/-) embryos(E14.5) and cultured with/without NELL-1(2000ng/ml) for 5 days. At cellular and molecular aspects, Primary embryonic mesenchymal progenitor cells(E11.5) of WT or Runx2(-/-) underwent chondrogenesis in pellet culture, treated with/without NELL-1(2000ng/ml), and collected at 7 and 21 days. Gene expression of Nell-1, chondrogenic and hypertrophic markers were performed by qPCR. HE, Safarin O and immunohistochemical staining were applied with morphological analyses. Results: Phenotypically, Nell-1 rescues Runx2(-/-) chondrocyte maturation in vivo, which can be observed by skeletal and Collagen X staining. NELL-1 can also promote the collagen X and Osteocalcin expression in the ex vivo cultured Runx2(-/-) limb buds immunohistochemically. For in vitro at day7, the down-regulation of Nell-1 and the delayed chondrogenic differentiation caused by Runx2 deficiency was partially rescues by NELL-1, which were evidenced by Collagen II, Aggrecan, Sox9 gene expression, and Safarin O, Collagen II staining. At day21, delayed chondrocytes hypertrophy caused by Runx2 deficiency was partially rescued by NELL-1. NELL-1 treatment increased Adamts4, Mmp13 and Ihh gene expression while it had no effect on Ocn and Alp, which were confirmed by histology. Conclusion: Our data demonstrate that NELL-1, a key functional mediator of Runx2 in osteogenesis, also functions as critical downstream mediator of Runx2 in chondrogenesis at both early and late stages using Runx2 deficient model.
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objectives: NELL-1 is a potent cytokine with pro-osteogenic and anti-osteoclastic effects. Specifically, it activates Wnt/β-catenin signaling and has been shown in multiple animal models to induce robust bone formation locally and systemically. Our previous systemic administration study using unmodified NELL-1 has shown successful increase of bone quality/quantity in both healthy and osteoporotic mice. However, an impractical q2d injection frequency limits NELL-1’s translation into a clinical application. For this reason, this study investigates the potential of PEGylation in improving NELL-1’s pharmacokinetics, while maintaining its bioactivity and osteogenic potential. Methods: First, we evaluated three PEGylation patterns(5K-linear, 20K-linear, and 40K-branched) for 1]NELL-1 thermostability via differential scanning fluorimetry(DSF), 2]half-life by using FITC conjugated NELL-1, and 3]in vitro osteogenic potential via mineralization studies in osteoblast cell line(Saos2). Next, we investigated the capacity of systemically administered PEG-NELL-1(1.25mg/kg, q4d) to promote bone formation in vivo, in 3-months-old CD-1 mice(n=10). To monitor bone mineral density(BMD), dual-energy X-ray absorptiometry(DXA) was performed weekly, with regions-of-interest(ROI) being distal femurs. Animals were sacrificed at 4 weeks post-treatment, and were analyzed by micro-Computed Tomography(microCT), histology, and immunohistochemistry. Results: PEGylation of NELL-1 was observed to enhance protein thermostability from 49.75oC up to 63.42oC(27% increase) and increase half-life from 5.5 hours to 16.1-31.3 hours(up to 6 fold), while retaining in vitro protein bioactivity levels comparable to unmodified NELL-1. In addition, systemic PEG-NELL-1 administration in vivo showed DXA result of a gradual and significant, 15.8% increase in femoral BMD compared to pre-treatment values. Post-mortem microCT also showed significant increases in trabecular and cortical bone BMD and volumetric measurements in the femurs. Conclusion: PEGylation of NELL-1 significantly increases protein thermostability and half-life, while retaining NELL-1’s osteogenic potential in vitro and in vivo. These exciting findings present PEGylated NELL-1 as a novel platform technology for systemic treatment of various osteodeficient disorders including osteoporosis and dysostosis syndromes.
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objective: Perivascular Stem Cells(PSC) are a novel stem cell population of pericytes(CD45-CD146+CD34-) and adventitial cells(CD45-CD146-CD34+). Unlike traditional stem cell populations, PSC do not require ex-vivo culture and are readily isolatable by FACS(fluorescent-activated cell sorting), therefore minimizing the risk of contamination and immunogenicity. NELL-1 is a cytokine, previously documented to promote robust bone formation. Here, we first investigate the potential of PSC in a small animal model, and second assess the potential of PSC±NELL-1 treatment to induce sturdy spinal fusion/fixation in a large animal, canine model. Method: First, athymic rats were randomly divided into groups of escalating PSC dosage, and implanted bilaterally with human PSC(hPSC) delivered upon a demineralized bone matrix(DBX) scaffold. Fusion was assessed four-weeks post-implantation by manual palpation, radiography, and immunohistochemistry(IHC). Next, we translated our findings into a canine model. Beagle dogs(n=6) were randomly distributed into three treatment groups to undergo spinal fusion: 1]Control(PBS), 2]canine PSC(cPSC), and 3]cPSC+NELL-1.Autologous cPSC were harvested, purified and re-implanted with or without the addition of NELL-1. Result: In rats, 80-100% spinal fusion was observed compared to 20% in control-treated animals. In hPSC-treated samples, the fractional bone volume,(bone volume/tissue volume, BV/TV) was statistically improved compared to the acellular control group. In addition, hPSC-treated samples exhibited increased trabecular thickness (Tb. Th) and significant, dose-dependent, increases in the trabecular bone number (Tb. N) In the large animal, canine model, control- and cPSC-treated animals achieved fusion rates of 0% and 50%, respectively. Remarkably, cPSC+NELL-1 treatment successfully induced 100% fusion by four-weeks post-implantation. Conclusion: Perivascular stem cells are capable of inducing robust spinal fixation in both small and large animal models.. Furthermore, combination treatment with PSC+NELL-1 is a superior treatment modality. In aggregate, these findings hold great clinical promise for PSC+NELL-1 combination treatment in future clinical application in orthopaedic and craniomaxillofacial surgical settings.
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objectives: Phenamil is a small, non-toxic derivative of Amiloride (FDA-approved potassium-sparing diuretic) previously shown to increase osteogenesis in vitro via down-regulation of BMP2 (bone morphogenetic protein 2) antagonist Smurf1 (Smad ubiquitylation regulatory factor-1). Furthermore, Phenamil has been observed to play a role in the balance of osteogenesis and adipogenesis via Trb-3(Tribbles homolog 3)-dependent induction of BMP signaling. Here, we evaluate the in vivo efficacy of orally administered Phenamil in preventing ovariectomy(OVX)-induced osteoporosis in mice. Methods: 10-week old CD1 mice (n=40) underwent either OVX or Sham surgery, followed by supplementation with Phenamil (50 mg/kg diet) or control chow. Bone mineral density (BMD) was evaluated weekly by DXA (dual-energy X-ray absorptiometry), with the distal femur and lumbar spine as the region of interest (ROI). Animals were sacrificed at four weeks post-operative for further analysis via micro-Computed Tomography (microCT), histology, histomorphometry, and immunohistochemistry for osteocalcin (OCN), alkaline phosphatase (ALP), and peroxisome proliferator-activated receptor gamma (PPARγ). Results: As anticipated, DXA results of ovariectomized, chow-fed constrol animals exhibited a significant decrease in mean BMD (10.7%) at four weeks. Ovariectomized animals supplemented with Phenamil, however, maintained a BMD comparable to Sham-operated, chow-fed animals. Moreover, Phenamil-fed, Sham-operated animals exhibited an 11-25% increase in BMD over four weeks. These findings were confirmed by microCT 3D reconstructions and quantifications, revealing significant increases in fractional bone volume (BV/TV) in animals supplemented with Phenamil. Bone formation was confirmed by histomorphometric measurements for bone area, trabecular thickness, and trabecular spacing. Immunohistochemical analysis demonstrated increased staining for OCN and ALP along bone perimeter in Phenamil-fed animals. Conclusion: Phenamil is a novel anabolic agent capable of preventing osteoporotic bone loss, while promoting bone formation in non-osteoporotic animals. As Phenamil is derived from FDA-approved Amiloride, which has shown per os efficacy and persistence through first-pass metabolism, these findings are highly translatable to clinical applications.
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objective: NELL-1 is an osteoinductive cytokine with known roles in craniofacial development. Recently, our laboratory has implicated Nell-1 in the pathoetiology of osteoporosis utilizing Nell-1 haploinssuficient mice, which prematurely developed an osteoporotic phenotype, with marked decreases in osteoblast:osteoclast(OB:OC) ratio, and reduced Wnt/β-catenin signaling. Here, we sought to further investigate the role of NELL-1 in bone maintenance and formation. Method: We first assessed the expression of NELL-1 in aging mice using qRT-PCR(quantitative reverse transcription polymerase chain reaction) and immunohistochemistry(IHC) for anti-NELL-1. Next, we evaluated the potential of systemically administered NELL-1 to reverse ovariectomy(OVX)-induced osteoporotic bone loss. Five weeks after undergoing OVX or Sham surgery, intravenous therapy was instituted(PBS or NELL-1, 1.25mg/kg q48hrs for 1mo). Progress was monitored weekly by dual-energy X-ray absorptiometry(DXA) scans for bone mineral density(BMD). We next evaluate the role of Integrinβ1 in NELL-1 signaling. To do so, mesenchymal stem cells(MSC) and pre-osteoclast cell-lines were treated with NELL-1±Integrinβ1 siRNA. Result: qRT-PCR and IHC quantifications revealed that Nell-1 expression is inversely proportional with skeletal aging. In our intravenous NELL-1 administration model, we observed that NELL-1 treatment gradually and significantly increased BMD in both non-osteoporotic and osteoporotic mice by a mean 13.9% compared to pre-treatment BMD. Immunohistochemical staining for osteocalcin(OCN) and tartrate-resistant acid phosphatase(TRAP) revealed that NELL-1 significantly increased the OB:OC ratio along bone perimeters. Lastly, it was observed that NELL-1 treatment significantly increased Wnt/β-catenin signaling in MSC and pre-osteoclasts. This signaling was inhibited when Integrinβ1 siRNA was incorporated, indicated NELL-1 activates the Wnt/β-catenin signaling via binding to Integrinβ1. Conclusion: These findings reveal that Nell-1 expression significantly decreases with skeletal aging. Further, NELL-1 intravenous administration can promote bone formation in both non-osteoporotic and osteoporotic mice via Integrinβ1 signaling. In aggregate, our findings suggest that systemic NELL-1 administration may hold clinical promise for future application in the treatment of osteoporotic bone loss.
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • [Show abstract] [Hide abstract]
    ABSTRACT: NELL1 is a large oligomeric secretory glycoprotein that functions as an osteoinductive factor. NELL1 contains several conserved domains, has structural similarities to thrombospondin-1, and supports osteoblastic cell adhesion through integrins. To define the structural requirements for NELL1-mediated cell adhesion, we prepared a series of recombinant NELL1 proteins (intact, deleted, and cysteine mutant) from a mammalian expression system and tested their activities. Deletion analysis demonstrated that the C-terminal cysteine-rich region of NELL1 is critical for cell adhesion activity of NELL1. Reducing agent treatment decreased the cell adhesion activity of full-length NELL1 but not of its C-terminal fragments, suggesting that the intramolecular disulfide bonds within this region are not functionally necessary, but other disulfide linkages in the N-terminal region of NELL1 may be involved in cell adhesion activity. By replacing cysteine residues with serines around the coiled-coil domain of NELL1 which is responsible for oligomerization, we created a mutant NELL1 protein that was unable to form homo-oligomers, and this monomeric mutant showed substantially lower cell adhesion activity than intact NELL1. These results suggest that an oligomerization-induced conformational change in the C-terminal region of NELL1 is important for the efficient mediation of cell adhesion and spreading by NELL1.
    Journal of Biological Chemistry 02/2014; · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fibromodulin (FMOD) is a small leucine-rich proteoglycan required for scarless fetal cutaneous wound repair. Interestingly, increased FMOD levels have been correlated with decreased transforming growth factor (TGF)-β1 expression in multiple fetal and adult rodent models. Our previous studies demonstrated that FMOD-deficiency in adult animals results in delayed wound closure and increased scar size accompanied by loose package collagen fiber networks with increased fibril diameter. In addition, we found that FMOD modulates in vitro expression and activities of TGF-β ligands in an isoform-specific manner. In this study, temporospatial expression profiles of TGF-β ligands and receptors in FMOD-null and wild-type (WT) mice were compared by immunohistochemical staining and quantitative reverse transcriptase-polymerase chain reaction using a full-thickness, primary intention wound closure model. During the inflammatory stage, elevated inflammatory infiltration accompanied by increased type I TGF-β receptor levels in individual inflammatory cells was observed in FMOD-null wounds. This increased inflammation was correlated with accelerated epithelial migration during the proliferative stage. On the other hand, significantly more robust expression of TGF-β3 and TGF-β receptors in FMOD-null wounds during the proliferative stage was associated with delayed dermal cell migration and proliferation, which led to postponed granulation tissue formation and wound closure and increased scar size. Compared with WT controls, expression of TGF-β ligands and receptors by FMOD-null dermal cells was markedly reduced during the remodeling stage, which may have contributed to the declined collagen synthesis capability and unordinary collagen architecture. Taken together, this study demonstrates that a single missing gene, FMOD, leads to conspicuous alternations in TGF-β ligand and receptor expression at all stages of wound repair in various cell types. Therefore, FMOD critically coordinates temporospatial distribution of TGF-β ligands and receptors in vivo, suggesting that FMOD modulates TGF-β bioactivity in a complex way beyond simple physical binding to promote proper wound healing.
    PLoS ONE 01/2014; 9(3):e90817. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Osteoporosis is a progressive bone disease due to low osteoblast activity and/or high osteoclast activity. NELL-1 is a potential therapy for osteoporosis because it specifically increases osteoblast differentiation. However, similar to other protein drugs, the bioavailability of NELL-1 may be limited by its in vivo half-life and rapid clearance from body. The purpose of the present study is to prolong NELL-1 circulation time in vivo by PEGylation with three monomeric PEG sizes (5, 20, 40 kDa). While linear PEG 5k yielded the most efficient PEGylation and the most thermally stable conjugate, linear PEG 20k resulted in the conjugate with the highest Mw and longest in vivo circulation. Compared to non-modified NELL-1, all three PEGylated conjugates showed enhanced thermal stability and each prolonged the in vivo circulation time significantly. Furthermore, PEGylated NELL-1 retained its osteoblastic activity without any appreciable cytotoxicity. These findings motivate further studies to evaluate the efficacy of PEGylated NELL-1 on the prevention and treatment of osteoporosis.
    Biomaterials 01/2014; 35(24):6614–6621. · 8.31 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Bone-morphogenetic protein 2 (BMP2) is currently the only FDA-approved osteoinductive growth factor used in clinical settings for bone regeneration and repair. However, the use of BMP2 is encumbered by numerous clinical complications, including postoperative inflammation and life-threatening cervical swelling. Thus, methods to prevent BMP2-induced inflammation would have far-reaching clinical implications toward improving current BMP2-based methods for bone regeneration. For the first time, we investigate the potential role of the growth factor NELL-1 in inhibiting BMP2-induced inflammation. Adult rats underwent a femoral bone onlay procedure, treated with either BMP2 protein (4 mg/ml), NELL-1 protein (4 mg/ml), or both proteins combined. Animals were evaluated at 3, 7, and 14 days postoperatively by histology, histomorphometry, immunohistochemistry and real time PCR for markers of inflammation (TNFα, IL6). The relative levels of TNFα and IL6 in serum were also detected by ELISA. The mechanism for NELL-1's anti-inflammatory effect was further assessed through examining inflammatory markers and generation of reactive oxygen species in the mouse embryonic fibroblast NIH3T3 cells. BMP2 significantly induced local inflammation, including an early and pronounced polymorphonuclear cell infiltration accompanied by increased expression of TNFα and IL6. Treatment with NELL-1 alone elicited no significant inflammatory response. However, NELL-1 significantly attenuated BMP2-induced inflammation by all markers and at all timepoints. These local findings were also confirmed using systemic serum inflammatory biomarkers (TNFα, IL6). In each case, NELL-1 fully reversed BMP2-induced systemic inflammation. Lastly, our findings were recapitulated in vitro, where NELL-1 suppressed BMP2 induced expression of inflammatory markers, as well as NF-κB transcriptional activity and generation of reactive oxygen species. BMP2-induced inflammation is a serious public health concern with potentially life-threatening complications. In the present study, we observed that the growth factor, NELL-1, significantly attenuates or completely reverses BMP2-induced inflammation. The mechanisms of NELL-1's anti-inflammatory effect are only partially elucidated, and may include reduction of NF-κB transcriptional activity or reactive oxygen species generation.
    Tissue Engineering Part A 06/2013; · 4.64 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Fibromodulin (FMOD) is an extracellular matrix (ECM) small leucine-rich proteoglycan (SLRP) that plays an important role in cell fate determination. Previous studies revealed that not only is FMOD critical in fetal-type scarless wound healing, but it also promotes adult wound closure and reduces scar formation. In addition, FMOD-deficient mice exhibit significantly reduced blood vessel regeneration in granulation tissues during wound healing. In this study, we investigated the effects of FMOD on angiogenesis, which is an important event in wound healing as well as embryonic development and tumorigenesis. We found that FMOD accelerated human umbilical vein endothelial HUVEC-CS cell adhesion, spreading, actin stress fiber formation, and eventually tube-like structure (TLS) network establishment in vitro. On a molecular level, by increasing expression of collagen I and III, angiopoietin (Ang)-2, and vascular endothelial growth factor (VEGF), as well as reducing the ratio of Ang-1/Ang-2, FMOD provided a favorable network to mobilize quiescent endothelial cells to an angiogenic phenotype. Moreover, we also confirmed that FMOD enhanced angiogenesis in vivo by using an in ovo chick embryo chorioallantoic membrane (CAM) assay. Therefore, our data demonstrate that FMOD is a pro-angiogenic and suggest a potential therapeutic role of FMOD in the treatment of conditions related to impaired angiogenesis.
    Biochemical and Biophysical Research Communications 06/2013; · 2.28 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Proceedings of the National Academy of Sciences 05/2013; · 9.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objectives: Autologous bone is the current gold standard of care for orthopaedic and craniofacial bone grafting procedures. However, its use is complicated by limited bone supply, extended surgical time, and donor site morbidity. The search for a novel source of osteoprogenitor cells has led to increased interest in mesenchymal stem cells (MSCs) for bone regeneration. In this study, we performed rat spinal fusions to evaluate the osteogenic potential of human Perivascular stem cells (hPSCs), a FACS-sorted, adipose-derived, homogenous MSC population. Methods: hPSCs were obtained after digestion of human lipoaspirates followed by FACS-sorting for ‘pericytes’ (CD146+CD34-CD45-) and ‘adventitial cells’ (CD146-CD34+CD45-). 22 athymic rats were randomly assigned to four treatment groups of increasing dosage of hPSCs (0, 0.15 million, 0.50 million, and 1.5 million cells) using a scaffold of a demineralized bone matrix (DBX). Animals underwent posterolateral spinal fusion of L4/L5 vertebrae and were sacrificed at four weeks postoperative. Fusion was assessed by manual palpation, microCT, histology, immunohistochemistry, and biomechanical analyses. Results: All hPSC treatment groups exhibited increased spinal fusion efficacy in comparison to acellular DBX treatment. Manual palpation revealed 80-100% fusion in hPSC-treated animals in comparison to 20% fusion in acellular DBX treatment. MicroCT analysis showed 138-140% increase in fractional Bone Volume (BV/TV) and 119-126% increase in Trabecular Number (Tb. N) with PSC treatment in comparison to acellular DBX. Histology and immunohistochemistry exhibited increased bone formation and vascularization, while biomechanical assays suggested increased stability when comparing hPSC treatment to acellular DBX. Conclusions: Here, we demonstrated the robust osteogenic potential of hPSCs in a rat spinal fusion model. hPSCs are an optimal stem cell population due to their abundant source and unrequired culture period, hence reducing the chance of contamination and immunogenic response. Although furtherer investigation is necessary, the present study shows promise for PSC-based products as an alternative for bone-graft procedures.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objectives: Treatment of cleft lip and palate requires multiple surgeries from birth through adulthood and frequent outpatient care. One of the critical surgeries occurs between ages 9 to 12, where an alveolar cleft bone graft is placed after the majority of maxillary growth is finished. Secondary alveolar bone grafts however can undergo a significant amount of resorption, often necessitating another graft surgery. Bisphosphonates (BPs) are a class of drugs which inhibit osteoclasts and can be used to control resorption. The aim of this pilot study was to investigate the effects of BPs on the success of bone grafts placed in alveolar defects in rats. Methods: Cancellous bone was harvested from the iliac crest and femur of an Inbred Sprague-Dawley rat. Recipient Inbred Sprague-Dawley rats underwent surgical creation of an alveolar defect and were divided into three groups. The control group (n=3) was given a subcutaneous injection of saline. The experimental group (n=3) was given a subcutaneous injection of the BP, Zoledronate (ZA) 1-week post-surgery at a concentration of 0.1 mg/kg. A negative control group (n=2) had an alveolar defect placed but were not given bone grafts. The rats were euthanized at 6 weeks and analysis of the alveolar defect was determined by micro-computed tomography (MicroCT) and histological analysis. Results: MicroCT analysis showed significantly more bone volume fraction (BV/TV) in the BP group (75.3 +/- 6.8%) compared to the Control group (43.1 +/- 9.1%) (p < 0.05). Histological analysis demonstrated bone graft incorporation with decreased osteoclast activity in the ZA treated group. Conclusions: ZA can be considered a therapeutic option to improve alveolar bone graft incorporation. A study with a larger sample size will be performed to confirm results. Future studies will focus on dosage and time-dependent effects of ZA on bone grafting.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
  • [Show abstract] [Hide abstract]
    ABSTRACT: Objectives: While microCT-based analysis is a powerful tool to validate micro-scale analysis and quantification in bone biology studies, it is also a highly user-sensitive technique that could introduce examiner bias, mostly in the process of ROI(region-of-interest) designation. As an added effort to standardize the method of microCT-based analysis, this study presents a new method of analyzing one of the most commonly used models; bone regeneration study using osteoporotic rat femoral model that presents a mixture of both bone resorption and apposition. Materials & Methods: A high-resolution microCT scanner, Skyscan 1172, was used in this study. Reconstruction, reorientation, and examination of scans were performed using NRecon, DataViewer, CTAn, and CTVol softwares provided by the manufacturer. Six 10-month old Sprague-Dawley rats were ovariectomized and given simultaneous injection of osteogenic protein NELL-1 in one femur. Contralateral femur was used as internal control. Eight weeks post-operation, femurs were harvested and microCT-scanned at 27.4 micrometer resolution under radiation source of 55 kV and 181 uA with a 0.5mm aluminum filter. MicroCT sections in transaxial, sagittal, and coronal planes were taken for direct comparison of right and left femurs by 3D color-coded superimposition using photoshop GS. Results: The study newly emphasizes the use of the contralateral femurs as internal control to determine relative bone apposition(Fig1.A) or resorption(Fig1.B) in experimental femurs, as similar porosity patterns will appear in both cases. 3D superimposition of both femurs verified the state of either augmentation or resorption of bone(Fig2.A-C), and enabled accurate designation of VOI(volume-of-interest)(Fig2.D). Conclusion: Assignment of an internal control femur and 3D superimposition of both femurs enables accurate and reproducible designing of VOI in microCT-based analysis of regenerative bone in osteoporotic rodent model. This new method of analysis is hoped to contribute to standardization of the use of microCT.
    03/2013
  • [Show abstract] [Hide abstract]
    ABSTRACT: An ideal mesenchymal stem cell (MSC) source for bone tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in phenotype and differentiation potential. PSCs consist of two distinct cell populations: [1] pericytes (CD146+, CD34-, CD45-), which surround capillaries and microvessels, and [2] adventitial cells (CD146-, CD34+, CD45-), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic bone model to develop the translational potential of our original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue. We evaluated human PSC (hPSC)-mediated bone formation and vascularization in vivo. We also examined the effects of hPSCs when combined with the novel craniosynostosis-associated protein, NELL-1 (Nel-like Molecule I). Implants consisting of demineralized bone matrix putty combined with either NELL-1 (3µg/µL), hPSC (2.5 x 105 cells), or hPSC+NELL-1 were inserted in the bicep femoris of SCID mice. Bone growth was evaluated using micro computed tomography, histology and immunohistochemistry over 4 weeks. Results demonstrated the osteogenic potential of hPSCs and the additive effect of hPSC+NELL-1 on bone formation and vasculogenesis. Comparable osteogenesis was observed with NELL-1 as compared to the more commonly used Bone Morphogenetic Protein (BMP)-2. Next, hPSCs induced greater implant vascularization than unsorted stromal vascular fraction (SVF) from patient-matched samples. Finally, we observed an additive effect on implant vascularization with hPSC+NELL-1 by histomorphometry and immunohistochemistry, accompanied by in vitro elaboration of vasculogenic growth factors. These findings hold significant implications for the cell/protein combination therapy hPSC+NELL-1 in the development of strategies for vascularized bone regeneration.
    Tissue Engineering Part A 02/2013; · 4.64 Impact Factor

Publication Stats

2k Citations
352.22 Total Impact Points

Institutions

  • 2007–2014
    • CSU Mentor
      Long Beach, California, United States
  • 1999–2014
    • University of California, Los Angeles
      • • School of Dentistry
      • • Department of Medicine
      • • Dental Research Institute
      • • Department of Bioengineering
      • • Department of Surgery
      • • Division of Plastic Surgery
      Los Angeles, California, United States
    • American Society of Ophthalmic Plastic and Reconstructive Surgery
      New York City, New York, United States
    • Niigata University
      • Brain Research Institute
      Niigata-shi, Niigata-ken, Japan
  • 2012
    • Shandong University
      Chi-nan-shih, Shandong Sheng, China
    • Indiana Orthopaedic Hospital
      Indianapolis, Indiana, United States
    • Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center
      Torrance, California, United States
  • 2011–2012
    • Nagoya University
      • Graduate School of Bio-Agricultural Sciences
      Nagoya-shi, Aichi-ken, Japan
    • Zhejiang University
      Hang-hsien, Zhejiang Sheng, China
    • Mount Sinai Medical Center
      New York City, New York, United States
  • 2010
    • Shanghai Jiao Tong University
      • Department of Orthopedics (Ninth People's Hospital)
      Shanghai, Shanghai Shi, China
  • 2008
    • New York University
      • Institute of Reconstructive Plastic Surgery
      New York City, NY, United States
  • 2005
    • Stanford University
      • Department of Surgery
      Stanford, CA, United States
  • 2004
    • University of Southern California
      Los Angeles, California, United States
  • 2000
    • CUNY Graduate Center
      New York City, New York, United States
    • Harbor-UCLA Medical Center
      Torrance, California, United States
  • 1993–1998
    • Harvard Medical School
      Boston, Massachusetts, United States