Osteoinductive Factors Research Articles

Investigation of low-level laser therapy potentiality on proliferation and differentiation of human osteoblast-like cells in the absence/presence of osteogenic factors

Several studies have shown that low-level laser irradiation (LLLI) has beneficial effects on bone regeneration. The objective of this study was to examine the in vitro effects of LLLI on proliferation and differentiation of a human osteoblast-like cell line (Saos-2 cell line). Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (659 nm; 10 mW power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated once or for three consecutive days with laser doses of 1 or 3 J/cm(2). The obtained results showed that laser stimulation enhances the proliferation potential of Saos-2 cells without changing their telomerase pattern or morphological characteristics. The effects on cell differentiation were assessed after three consecutive laser irradiation treatments in the presence or absence of osteo-inductive factors on day 14. Enhanced secretion of proteins specific for differentiation toward bone as well as calcium deposition and alkaline phosphatase activity were observed in irradiated cells cultured in a medium not supplemented with osteogenic factors. Taken together these findings indicate that laser treatment enhances the in vitro proliferation of Saos-2 cells, and also influences their osteogenic maturation, which suggest it is a helpful application for bone tissue regeneration.

Involvement of the Osteoinductive Factors, Tmem119 and BMP-2, and the ER Stress Response PERK–eIF2α–ATF4 Pathway in the Commitment of Myoblastic into Osteoblastic Cells

The osteoinductive factors BMP-2 and Tmem119 that promote the differentiation of myoblasts into osteoblasts, each increase the levels of the other. However, the relative contributions of BMP-2 and Tmem119 to the osteogenic differentiation and the mechanisms involved are incompletely understood. In the present study, we examined the relationship among BMP-2, Tmem119, and the PERK-eIF2α-ATF4 endoplasmic reticulum (ER) stress response pathway in the differentiation of C2C12 myoblasts into osteoblastic cells. Both BMP-2 and Tmem119 induced levels of the osteoblast markers Runx2, Osterix, Col1a1, ALP, and osteocalcin, as well as mineralization. BMP-2 activation of the ER stress sensor PERK stimulated phosphorylation of eIF2α and led to increased biosynthesis of the osteoblast differentiation factor ATF4. When dephosphorylation of eIF2α was blocked by the selective inhibitor salubrinal, the osteogenic effects of BMP-2 and Tmem119 were enhanced further. Although BMP-2 stimulated both P-eIF2α and ATF4 levels, Tmem119 had no effect on P-eIF2α but stimulated ATF4 only. Reduction in endogenous Tmem119 levels by siRNA reduced both basal and BMP-2-stimulated levels of the ATF4 protein. In conclusion, BMP-2 stimulates differentiation of myoblasts into osteoblasts via the PERK-eIF2α-ATF4 pathway but in addition stimulates Tmem119, which itself increases ATF4. Hence, BMP-2 stimulates ATF4 both dependently and independently of the PERK-eIF2α ER stress response pathway.

Evaluating apatite formation and osteogenic activity of electrospun composites for bone tissue engineering

Significant interest has been in examining calcium phosphate ceramics, specifically β-tricalcium phosphate (β-TCP) (Ca3 (PO4)2 ) and synthetic hydroxyapatite (HA) (Ca10 (PO4)6 (OH)2 ), in composites and more recently, in fibrous composites formed using the electrospinning technique for bone tissue engineering applications. Calcium phosphate ceramics are sought because they can be bone bioactive, which means an apatite forms on their surface that facilitates bonding to bone tissue, and are osteoconductive. However, studies examining the bioactivity of electrospun composites containing calcium phosphates and their corresponding osteogenic activity have been limited. In this study, electrospun composites consisting of (20/80) HA/TCP nanoceramics and poly (ϵ-caprolactone) (PCL) were fabricated. Solvent and solvent combinations were evaluated to form scaffolds with a maximum concentration and dispersion of ceramic and pore sizes large enough for cell infiltration and tissue growth. PCL was dissolved in either methylene chloride (Composite-MC) or a combination of methylene chloride (80%) and dimethylformamide (20%; Composite-MC + DMF). Composites were evaluated in vitro for degradation, apatite formation, and osteogenic differentiation of human mesenchymal stem cells (MSCs) with an emphasis on temporal gene expression of osteogenic markers and the pluripotent gene Sox-2. Apatite formation and the osteogenic differentiation was the greatest for Composite-MC as determined by gene expression, protein production and biochemical markers, even without the presence of osteoinductive factors in the media, in comparison to Composite-MC + DMF and unfilled PCL mats. Sox-2 levels also reduced over time. The results of this study demonstrate that the solvent or solvent combination used in preparing the electrospun composite mats plays a critical role in determining their bioactivity which may, in turn, affect cell behavior.

Complete subchondral bone defect regeneration with a tricalcium phosphate collagen implant and osteoinductive growth factors: A randomized controlled study in Göttingen minipigs

The restoration and reconstruction of osseous defects close to the joint, constitutes a challenging field for reconstructive surgery. A dual-layer implant of β-tricalcium phosphate (TCP) and a collagens I/III scaffold was evaluated in a prospective, randomized comparison in a larger animal model. For this purpose, a standardized osteochondral defect was created in the medial facet of the patellar groove in both stifle joints of Göttingen minipigs. Critical-size osseous defects were either left empty (spontaneous healing; group 1; n = 12) or treated with the two-layer TCP collagen implant (group 2; n = 12). In group 3 (n = 12), additional growth factor mixture (GFM) was supplemented (bone morphogenetic proteins 2, 3, 4, 6, 7, and TGF-β1, 2, 3). Osseous defect regeneration was assessed at 6, 12, and 52 weeks postoperatively (n = 4). Qualitative and quantitative histomorphometric assessment of defect regeneration and bone substitute resorption was conducted by means of light microscopy, fluorescence microscopy, and microradiography. Critical-size defects did not heal spontaneously throughout follow-up (group 1: max. 21.84 ± 2.81% defect area at 52 weeks). The TCP layer of the implant significantly increased the amount of new bone formation with 29.8 ± 9.68% at 6 weeks and 40.09 ± 4.76% at 12 weeks when compared with controls. After 52 weeks, the TCP was almost fully degraded (4.35 ± 3.70%) and the defect was restored with lamellar trabecular bone (31.28 ± 5.02%). Growth factor supplementation resulted in earlier resorption of the TCP implant and faster defect regeneration. The dual-layer TCP collagen implant is suitable to restore subchondral osseous defects. Additional use of GFM increased the resorption of the TCP layer, but did not foster new bone formation.

Segmental mandibular bone reconstruction with a carbonate‐substituted hydroxyapatite‐coated modular endoprosthetic poly(ɛ‐caprolactone) scaffold in Macaca fascicularis

A bio-degradable scaffold incorporating osteoinductive factors is one of the alternative methods for achieving the regeneration of a mandibular bone defect. The current pilot study addressed such a bone reconstruction in a non-human primate model, Macaca fascicularis monkeys, with an engineered poly(ɛ-caprolactone) (PCL) scaffold, provided with a carbonate-substituted hydroxyapatite coating. The scaffolds were implanted into unilaterally created mandibular segmental defects in 24 monkeys. Three experimental groups were formed: (1) scaffolds with rhBMP-2 (n = 8), (2) scaffolds with autologous mixed bone marrow cells (n = 8), and (3) empty scaffolds as a control group (n = 8). Evaluation was based on clinical observation as well as micro-CT, mechanical, and histological analyses. Despite a high infection rate, the overall results showed that the currently designed PCL scaffolds had insufficient load-bearing capability, and complete bone union was not achieved after 6 months of implantation. Nevertheless, the group of PCL scaffolds loaded with rhBMP-2 showed evidence of bone-regenerative potential, in contrast to PCL with autologous mixed bone marrow cells and the control group.

Characterization of scaffold carriers for BMP9‐transduced osteoblastic progenitor cells in bone regeneration

AbstractSuccessful bone tissue engineering at least requires sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We have demonstrated that BMP9 is one of the most potent factors in inducing osteogenic differentiation of mesenchymal progenitors. To facilitate the potential use of cell‐based BMP9 gene therapy for bone regeneration, we characterize the in vivo osteoconductive activities and bone regeneration potential of three clinically used scaffold materials, type I collagen sponge, hydroxyapatite‐tricalcium phosphate (HA‐TCP), and demineralized bone matrix (DBM), using BMP9‐expressing C2C12 osteoblastic progenitor cells. We find that recombinant adenovirus‐mediated BMP9 expression effectively induces osteogenic differentiation in C2C12 cells. Although direct subcutaneous injection of BMP9‐transduced C2C12 cells forms ectopic bony masses, subcutaneous implantation of BMP9‐expressing C2C12 cells with collagen sponge or HA‐TCP scaffold yields the most robust and mature cancellous bone formation, whereas the DBM carrier group forms no or minimal bone masses. Our results suggest that collagen sponge and HA‐TCP scaffold carriers may provide more cell‐friendly environment to support the survival, propagation, and ultimately differentiation of BMP9‐expressing progenitor cells. This line of investigation should provide important experimental evidence for further preclinical studies in BMP9‐mediated cell‐based approach to bone tissue engineering. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3429–3438, 2014.

Characterization of scaffold carriers for BMP9-transduced osteoblastic progenitor cells in bone regeneration

Successful bone tissue engineering at least requires sufficient osteoblast progenitors, efficient osteoinductive factors, and biocompatible scaffolding materials. We have demonstrated that BMP9 is one of the most potent factors in inducing osteogenic differentiation of mesenchymal progenitors. To facilitate the potential use of cell-based BMP9 gene therapy for bone regeneration, we characterize the in vivo osteoconductive activities and bone regeneration potential of three clinically used scaffold materials, type I collagen sponge, hydroxyapatite-tricalcium phosphate (HA-TCP), and demineralized bone matrix (DBM), using BMP9-expressing C2C12 osteoblastic progenitor cells. We find that recombinant adenovirus-mediated BMP9 expression effectively induces osteogenic differentiation in C2C12 cells. Although direct subcutaneous injection of BMP9-transduced C2C12 cells forms ectopic bony masses, subcutaneous implantation of BMP9-expressing C2C12 cells with collagen sponge or HA-TCP scaffold yields the most robust and mature cancellous bone formation, whereas the DBM carrier group forms no or minimal bone masses. Our results suggest that collagen sponge and HA-TCP scaffold carriers may provide more cell-friendly environment to support the survival, propagation, and ultimately differentiation of BMP9-expressing progenitor cells. This line of investigation should provide important experimental evidence for further preclinical studies in BMP9-mediated cell-based approach to bone tissue engineering.

Enhanced Osteogenesis of Adipose Derived Stem Cells with Noggin Suppression and Delivery of BMP-2

Bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors. However, BMPs are highly pleiotropic molecules and their supra-physiological high dose requirement leads to adverse side effects and inefficient bone formation. Thus, there is a need to develop alternative osteoinductive growth factor strategies that can effectively complement BMP activity. In this study, we intrinsically stimulated BMP signaling in adipose derived stem cells (ASCs) by downregulating noggin, a potent BMP antagonist, using an RNAi strategy. ASCs transduced with noggin shRNA significantly enhanced osteogenic differentiation of cells. The potency of endogenous BMPs was subsequently enhanced by stimulating ASCs with exogenous BMPs at a significantly reduced dose. The level of mineralization in noggin shRNA treated ASCs when treated with BMP-2 was comparable to that of control shRNA treated cell treated with 10-fold more BMP-2. The complementary strategy of noggin suppression + BMP-2 to enhance osteogenesis was further confirmed in 3D in vitro environments using scaffolds consisting of chitosan (CH), chondroitin sulfate (CS), and apatite layer on their surfaces designed to slowly release BMP-2. This finding supports the novel therapeutic potential of this complementary strategy in bone regeneration.

Evaluation of Injectable Constructs for Bone Repair with a Subperiosteal Cranial Model in the Rat

While testing regenerative medicine strategies, the use of animal models that match the research questions and that are related to clinical translation is crucial. During the initial stage of evaluating new strategies for bone repair, the main goal is to state whether the strategies efficiently induce the formation of new bone tissue at an orthotopic site. Here, we present a subperiosteal model in rat calvaria that allow the evaluation of a broad range of approaches including bone augmentation, replacement and regeneration. The model is a fast to perform, minimally invasive, and has clearly defined control groups. The procedure enables to evaluate the outcomes quantitatively using micro-computed tomography and qualitatively by histology and immunohistochemistry. We established this new model, using bone morphogenetic protein-2 as an osteoinductive factor and hyaluronic acid hydrogel as injectable biomaterial. We showed that this subperiosteal cranial model offers a minimally invasive and promising solution for a rapid initial evaluation of injectables for bone repair. We believe that this approach could be a powerful platform for orthopedic research and regenerative medicine.

2011 AOA Symposium: Tissue Engineering and Tissue Regeneration

Improvements in our understanding of biological and biomechanical aspects of connective-tissue degeneration, healing, and repair have revealed the potential to develop novel approaches to tissue repair and regeneration. New knowledge about the basic cellular and molecular processes that govern tissue repair has emerged from the fields of cell biology, molecular biology, and molecular genetics. Ongoing developments in related fields, such as biomaterials and stem cells, have also suggested new ways to attack old problems in orthopaedics. This symposium brought together experts to address several important areas: (1) the use of local osteoinductive factors for spine fusion, (2) systemic agents and mesenchymal stromal cells to manipulate bone formation and remodeling, and (3) the use of scaffolds and platelet-derived factors for ligament-healing. We discuss biologic approaches to tissue-healing and regeneration, ranging from experimental techniques that are currently in preclinical testing to techniques that are currently in use. The basic principles related to tissue-healing and regeneration presented here will provide the basis for future studies in tissue engineering and related fields. Spine arthrodesis is one of the most challenging applications for bone graft materials and bone-healing. The nonunion rate associated with arthrodesis for management of degenerative lumbar spine disease ranges from 10% to 45%1,2. The combination of biologic and environmental factors makes the achievement of spine fusion sensitive to systemic issues that inhibit bone-healing, such as nonsteroidal anti-inflammatory drugs, corticosteroids, and nicotine. Bone graft substitutes are associated with one or more of four potential healing mechanisms3. An osteogenic material, by definition, has live osteoblasts that are capable of making bone matrix, which mineralizes normally. Osteoconductivity is the ability of a material to serve as a scaffold to support new bone ingrowth and/or ongrowth and can be influenced by the three-dimensional macrostructure as well as features such as …

BMP2-Induced Inflammation Can Be Suppressed by the Osteoinductive Growth Factor NELL-1

Bone-morphogenetic protein 2 (BMP2) is currently the only Food and Drug Administration-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 Nel-like molecule-1 (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 (ROS) 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 ROS. 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 ROS generation.

NELL-1 based demineralized bone graft promotes rat spine fusion as compared to commercially available BMP-2 product

BackgroundSpinal fusion is among the most commonly performed orthopaedic procedures. Unfortunately, current treatments such as autologous bone grafting or recombinant proteins (BMP-2) have numerous clinical shortcomings. Here, we directly compare the efficacy of NELL-1, a novel osteoinductive growth factor, to two currently available treatments, (1) recombinant BMP-2 and (2) iliac crest bone grafting, in a spinal fusion model. MethodsTwenty-six skeletally mature athymic rats underwent posterolateral spine fusion of L4/L5 vertebrae. Treatment groups included NELL-1 (10 and 50μg) in a demineralized bone matrix (DBX), as compared to BMP-2 (90μg) in an absorbable collagen sponge (ACS) or morselized iliac crest bone. Scaffolds without recombinant protein were used as controls. Animals were sacrificed at 4weeks post-operative and fusion was assessed by manual palpation, radiography [high-resolution X-ray, micro-computed tomography (microCT)], histology (hematoxylin and eosin, Masson’s trichrome) and immunohistochemistry (osteocalcin). ResultsResults showed 100% fusion in all NELL-1- and BMP-2-treated samples. In contrast, lower rates of fusion were observed in scaffold-only and bone graft treatment groups. MicroCT scans revealed radiographic evidence of fusion among spines treated with NELL-1. Bone bridging was also observed with BMP-2 treatment, but was accompanied by inner radiolucency, suggesting cyst-like bone formation. Histologically, NELL-1-treated grafts showed increased bone formation, endochondral ossification and vascularization. Although BMP-2 treated grafts exhibited increased bone formation and angiogenesis, numerous adipocytes were also observed. ConclusionNELL-1-based bone grafts are comparable to BMP-2+ACS in spinal fusion efficacy. Histological differences were observed however, including robust endochondral ossification with NELL-1 treatment as compared to lipid-filled bone with BMP-2 treatment. These findings suggest NELL-1 based bone grafts show promise for future efforts in skeletal tissue engineering.

In Vitro Study of a Novel Oxysterol for Osteogenic Differentiation on Rabbit Bone Marrow Stromal Cells

Bone morphogenetic proteins (BMPs) are powerful osteoinductive growth factors but are associated with exorbitant costs and undesirable side effects. Oxysterols are biocompatible cholesterol oxidation products with osteoinductive properties that may represent an alternative to BMP. In this study, the authors examine the osteogenic potential and mechanisms of actions of oxysterol 49, a novel oxysterol analogue, in primary rabbit bone marrow stromal cells. Bone marrow stromal cells were isolated from the iliac crests of New Zealand White rabbits and then treated with various concentrations of oxysterol 49 or BMP-2, either alone or in combination. Alkaline phosphatase activity and expression of osteocalcin and osteopontin were evaluated. The effect of treatment of cells with cyclopamine, a known hedgehog signaling pathway inhibitor, was also assessed. Alkaline phosphatase activity was increased in cells treated with 1 µM oxysterol 49 relative to cells treated with BMP-2. Expression of osteocalcin and osteopontin in cells treated with oxysterol 49 and BMP-2 was equivalent. Alkaline phosphatase activity was decreased with the addition of cyclopamine. Combined treatment with oxysterol 49 and BMP-2 resulted in additive increases in alkaline phosphatase activity and osteocalcin and osteopontin expression. Oxysterol 49 has osteoinductive properties that are similar to those of BMP-2 in rabbit bone marrow stromal cells. The mechanism of this activity is at least in part related to the hedgehog signaling pathway. The two growth factors demonstrate additive effects when used in combination. Further study is required to examine the potential role of oxysterol 49 as a complement or alternative to BMP-2 in bone tissue engineering.

Bone tissue engineering with bone marrow-derived stromal cells integrated with concentrated growth factor in Rattus norvegicus calvaria defect model

Concentrated growth factor (CGF) is an autologous leukocyte-rich and platelet-rich fibrin (L-PRF) biomaterial termed "second-generation platelet concentrate". CGF contains autologous osteoinductive platelet growth factors and an osteoconductive fibrin matrix. The purpose of this study was to assess the ability of CGF combined with bone marrow stromal cells (BMSCs) to heal critical-size rat calvaria defects in vivo and to modulate the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. In the in-vivo study, the CGF group regenerated bone better than the control group, and combined therapy with CGF and BMSCs almost completely repaired critical-size bone defects within 12 weeks after surgery. In the in-vitro study, the CGF extract, at concentrations between 1 and 10%, promoted proliferation, osteogenic maturation, and mineralization of hTERT-E6/E7 human MSCs in a dose-dependent manner but had an inhibitory effect at higher concentrations. In conclusion, a CGF extract promoted the proliferation, osteogenic maturation, and mineralization of mesenchymal stem cells in vitro, and combination therapy with CGF and BMSCs resulted in excellent healing of critical-size bone defects in vivo.

Enhanced reconstruction of long bone architecture by a growth factor mutant combining positive features of GDF-5 and BMP-2

Non healing bone defects remain a worldwide health problem and still only few osteoinductive growth factors are available for clinical use in bone regeneration. By introducing BMP-2 residues into growth and differentiation factor (GDF)-5 we recently produced a mutant GDF-5 protein BB-1 which enhanced heterotopic bone formation in mice. Designed to combine positive features of GDF-5 and BMP-2, we suspected that this new growth factor variant may improve long bone healing compared to the parent molecules and intended to unravel functional mechanisms behind its action. BB-1 acquired an increased binding affinity to the BMP-IA receptor, mediated enhanced osteogenic induction of human mesenchymal stem cells versus GDF-5 and higher VEGF secretion than BMP-2 in vitro. Rabbit radius defects treated with a BB-1-coated collagen carrier healed earlier and with increased bone volume compared to BMP-2 and GDF-5 according to in vivo micro-CT follow-up. While BMP-2 callus often remained spongy, BB-1 supported earlier corticalis and marrow cavity formation, showing no pseudojoint persistence like with GDF-5. Thus, by combining positive angiogenic and osteogenic features of GDF-5 and BMP-2, only BB-1 restored a natural bone architecture within 12 weeks, rendering this promising growth factor variant especially promising for long bone regeneration.

Bioactive silicate nanoplatelets for osteogenic differentiation of human mesenchymal stem cells.

Novel silicate nanoplatelets that induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) in the absence of any osteoinductive factor are reported. The presence of the silicate triggers a set of events that follows the temporal pattern of osteogenic differentiation. These findings underscore the potential applications of these silicate nanoplatelets in designing bioactive scaffolds for musculoskeletal tissue engineering.

Orthopedic bioactive implants: Hydrogel enrichment of macroporous titanium for the delivery of mesenchymal stem cells and strontium

Insufficient implant stability is an important determinant in the failure of cementless prostheses. To improve osseointegration, we aim at generating a bioactive implant combining a macroporous titanium (TT) with a biocompatible hydrogel to encapsulate osteo-inductive factors and osteoprogenitor cells. Amidation and cross-linking degree of an amidated carboxymethylcellulose hydrogel (CMCA) were characterized by FT-IR spectrometry and mechanical testing. Bone marrow mesenchymal stem cells (BMSCs) from osteoarthritic patients were cultured on CMCA hydrogels, TT, and TT loaded with CMCA (TT + CMCA) with an optimized concentration of SrCl2 to evaluate cell viability and osteo-differentiation. Amidation and cross-linking degree were homogeneous among independent CMCA batches. SrCl2 at 5 μg/mL significantly improved BMSCs osteo-differentiation increasing calcified matrix (P < 0.01), type I collagen expression (P < 0.05) and alkaline phosphatase activity. TT + CMCA samples better retained cells into the TT mesh, significantly improving cell seeding efficiency with respect to TT (P < 0.05). BMSCs on TT + CMCA underwent a more efficient osteo-differentiation with higher alkaline phosphatase (P < 0.05) and calcium levels compared to cells on TT. Based on these in vitro results, we envision the association of TT with strontium-enriched CMCA and BMSCs as a promising strategy to generate bioactive implants promoting bone neoformation at the implant site.

Mesenchymal Stem Cells from Bichat's Fat Pad:In VitroComparison with Adipose-Derived Stem Cells from Subcutaneous Tissue

Adipose-derived stem/stromal cells (ASCs) are progenitor cells used in bone tissue engineering and regenerative medicine. Since Bichat's fat pad is easily accessible for dentists and maxillo-facial surgeons, we compared the features of ASCs from Bichat's fat pad (BFP-ASCs) with human ASCs from subcutaneous adipose tissue (SC-ASCs). BFP-ASCs isolated from a small amount of tissue were characterized for their stemness and multidifferentiative ability. They showed an important clonogenic ability and the typical mesenchymal stem cell immunophenotype. Moreover, when properly induced, osteogenic and adipogenic differentiation markers, such as alkaline phosphatase activity, collagen deposition and lipid vacuoles formation, were promptly observed. Growth of both BFP-ASCs and SC-ASCs in the presence of human serum and their adhesion to natural and synthetic scaffolds were also assessed. Both types of ASCs adapted rapidly to human autologous or heterologous sera, increasing their proliferation rate compared to standard culture condition, and all the cells adhered finely to bone, periodontal ligament, collagen membrane, and polyglycol acid filaments that are present in the oral cavity or are commonly used in oral surgery. At last, we showed that amelogenin seems to be an early osteoinductive factor for BFP-ASCs, but not SC-ASCs, in vitro. We conclude that Bichat's fat pad contains BFP-ASCs with stemness features that are able to differentiate and adhere to biological supports and synthetic materials. They are also able to proliferate in the presence of human serum. For all these reasons we propose BFP-ASCs for future therapies of periodontal defects and bone regeneration.

High affinity binding of an engineered, modular peptide to bone tissue.

Bone grafting procedures have become common due in part to a global trend of population aging. Native bone graft is a popular choice when compared to various synthetic bone graft substitutes, owing to superior biological activity. Nonetheless, the insufficient ability of bone allograft to induce new bone formation and the insufficient remodeling of native bone grafts call for osteoinductive factors during bone repair, exemplified by recombinant human bone morphogenetic protein 2 (rhBMP2). We previously developed a modular bone morphogenetic peptide (mBMP) to address complications associated with the clinical use of rhBMP2 as a bone graft substitute. The mBMP is designed to strongly bind to hydroxyapatite, the main inorganic component of bone and teeth, and to provide pro-osteogenic properties analogous to rhBMP2. Our previous in vivo animal studies showed that mBMP bound to hydroxyapatite-coated orthopedic implants with high affinity and stimulated new bone formation. In this study, we demonstrate specific binding of mBMP to native bone grafts. The results show that mBMP binds with high affinity to both cortical and trabecular bones, and that the binding is dependent on the mBMP concentration and incubation time. Importantly, efficient mBMP binding is also achieved in an ex vivo bone bioreactor where bone tissue is maintained viable for several weeks. In addition, mBMP binding can be localized with spatial control on native bone tissue via simple methods, such as dip-coating, spotting, and direct writing. Taken together with the pro-osteogenic activity of mBMP established in previous bone repair models, these results suggest that mBMP may promote bone healing when coated on native bone grafts in a clinically compatible manner.

Polyanhydride copolymer and bioceramic composites as bone substitutes

Background/PurposeDurable mechanical strength and biocompatibility are the two major requirements for osteogenic scaffolds. Polyanhydrides are a class of biodegradable polymers characterized by anhydride bonds that connect repeating units of the polymer backbone chain. Hydroxyapatite (HAP) is the main component of human bone and is a good osteoinductive factor that promotes bone mineralization. This work validates the combination of polyanhydrides and HAP for biomedical application. MethodsPolyanhydride copolymers were fabricated from sebacic acid (SA) and 1,6-bis(p-carboxyphenoxy)hexane (CPH). HAP was surface-modified by polycaprolactone (PCL), and testing tablets were made using different ratios of copolymers and surface-grafted HAP (g-HAP). Degradation tests were performed to evaluate mechanical strength, pH, and weight loss. Biocompatibility was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and live/dead stain test. Cell affinity was measured using scanning electronic microscopy (SEM). ResultsThe favorable surface erosion property of polyanhydrides prevented marked changes in the mechanical properties over time. In addition, the degradation byproducts of the copolymer did not cause a serious decline in pH and were less harmful to the cells. g-HAP increased cell affinity for the polymer surface. ConclusionThe research team synthesized polyanhydride/g-HAP composites with high mechanical strength, slow degradation, and excellent biocompatibility. The result showed that a CPH/SA ratio of 7:3 in combination with 10 wt% g-HAP was optimal as bone substitute.