Three-dimensional kagome structures in a PCL/HA-based hydrogel scaffold to lead slow BMP-2 release for effective bone regeneration

Osteoconductive function is remarkably low in bone disease in the absence of bone tissue surrounding the grafting site,or if the bone tissue is in poor condition.Thus,an effective bone graft in terms of both osteoconductivity and osteoinductivity is required for clinical therapy.Recently,the three-dimensional(3D)kagome structure has been shown to be advantageous for bone tissue regeneration due to its mechanical properties.In this study,a polycaprolactone(PCL)kagome-structure scaffold containing a hyaluronic acid(HA)-based hydrogel was fabricated using a 3D printing technique.The retention capacity of the hydrogel in the scaffold was assessed in vivo with a rat calvaria subcutaneous model for 3 weeks,and the results were compared with those obtained with conventional 3D-printed PCL grid-structure scaffolds containing HA-based hydrogel and bulk-type HA-based hydrogel.The retained hydrogel in the kagome-structure scaffold was further evaluated by in vivo imaging system analysis.To further reinforce the osteoinductivity of the kagome-structure scaffold,a PCL kagome-structure scaffold with bone morphogenetic protein-2(BMP-2)containing HA hydrogel was fabricated and implanted in a calvarial defect model of rabbits for 16 weeks.The bone regeneration characteristics were evaluated with hematoxylin and eosin(H&E),Masson’s trichrome staining,and micro-CT image analysis.

Influence of bone morphogenetic proteins-2 and strontium chloride on the human umbilical cord mesenchymal stem cells proliferation and osteogenic differentiation

Objective To study the effects of combination of bone morphogenetic protein-2 (BMP-2) and strontium chloride on proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells(hUCM-SCs)in vitro culture.

Chondrogenic Differentiation of Mouse Bone Marrow Mesenchymal Stem Cells Induced by Cartilage-derived Morphogenetic Protein-2 In Vitro

To study the cartilage differentiation of mouse mesenchymal stem cells （MSCs） induced by cartilage-derived morphogenetic proteins-2 in vitro, the MSCs were isolated from mouse bone marrow and cultured in vitro. The cells in passage 3 were induced into chondrogenic differentiation with different concentrations of recombinant human cartilage-derived morphogenetic proteins-2 （0, 10, 20, 50 and 100 ng/mL）. After 14 days of induction, morphology of cells was observed under phase-contrast microscope. Collagen Ⅱ mRNA and protein were examined with RT-PCR, Western blotting and immunocytochemistry respectively and the sulfate glycosaminoglycan was measured by Alcian blue staining. RT-PCR showed that CDMP-2 could promote expression of collagen Ⅱ mRNA in an dose-dependant manner, especially at the concentration of 50 ng/mL and 100 ng/mL. Immunocytochemistry and Western blotting revealed a similar change. Alcian blue staining exhibited deposition of typical cartilage extracellular matrix. Our results suggest that mouse bone marrow mesencymal stem cells can differentiate into chondrogenic phonotype with the induction of CDMP-2 in vitro, which provides a basis for further research on the role of CDMP-2 in chondrogenesis.

The Effect of Simvastatin on mRNA Expression of Transforming Growth Factor-β1,Bone Morphogenetic Protein-2 and Vascular Endothelial Growth Factor in Tooth Extraction Socket

Aim To determine the effect of local simvastatin application on the mRNA expression level of transforming growth factor-β1 （TGF-β1）, bone morphogenetic protein-2 （BMP-2） and vascular endothelial growth factor （VEGF） in the tooth sockets of rat. Methodology Forty-eight male Wistar rats were randomly divided into experimental and control groups （n=24）. Polylactic acid/polyglycolic acid copolymer carriers, with or without simvastatin, were implanted into extraction sockets of right mandibular incisors. The expression of TGF-β1, BMP-2 and VEGF mRNA was determined by in situ hybridization in the tooth extraction socket at five days, one week, two weeks and four weeks after implantation. Results The fusiform stroma cells in the tooth extraction socket began to express TGF-β1, BMP-2 and VEGF mRNA in both experimental and control groups from one week after tooth extraction until the end of experiment. The expression of TGF-131 and BMP-2 mRNA in the experimental group was significantly up-regulated after one, two and four weeks, and expression of VEGF mRNA was significantly increased after one and two weeks compared with that in the control group. Conclusion The findings indicate that local administration of simvastatin can influence alveolar bone remodeling by regulating the expression of a school of growth factors which are crucial to osteogenesis in the tooth extraction socket.

Osteogenesis and Degradation Behavior of rhBMP-27/β-Tricalcium Phosphate Porous Composite Materials

Ultrafine-tricalcium phosphate(β-TCP)powders with good crystalline structure were produced by a new process through bone tissue engineering approach rorous β-TCPcermic was combined with recombined human bone morphogenetic proteins-2(rhBMP-2)to develop a novel composite material ,osteogenesis capacity of the composite was investigated intramuscularly in rat with histological analyses and SEM examination pureβ-TCP porous carmic wsa investigated as the control results show that the compostie materials possess good bilcompatibility biodegradation and strong osteogenesis capacity through inductive process after implantation material degradation began from 2 weeks post-implantation accompanying with the changing o pore structure with the enwrapping and separation fo materials by hyperplatic mesenchymal cells and fibroblast and with the phagocytose reaction of multinucleated giant cells early in 72h immature cartilage could be found within novel composite mature lamellar bone was induced to generate after 3 weeks with strong osteoinduction capacity and controlable bildegradation the novel rhBMP-2\β-TCP porous ceramic is expected to be a promising bone grafting substitute for bone tissue engineering

Both Atorvastatin and Probucol Can Down-regulate BMP-2 Expression Induced by Oxidized Low Density Lipoprotein in Human Umbilical Vein Cells

Objectives Bone morphogenetic protein-2 (BMP-2) plays an key role both in vascular development and pathophysiological processes. However, the mechanisms of oxidized low density lipoprotein (ox-LDL) and combinated with atorvastatin or probucol on BMP-2 expression are entirely unknown in human umbilical vein cells. Methods The HUVECs were treated by ox-LDL and combinated with atorvastatin, probucol. The expression of BMP-2, NF-κB65, PPARγ mRNA was examined by RT-PCR analysis and ELISA method. The MDA and SOD were detected by routine methods. Results Ox-LDL can induced BMP-2 mNRA expression, associated with NF-κB65 mNRA expression activation. Both atorvastatin and probucol can suppress BMP-2 and NF-κB65 expression induced by oxLDL and upregulate the expression of PPARγ. Furthermore, the increase of supernatant MDA levels and decrease of supernatant SOD levels resulted from oxLDL treatment can be reversed by probucol or atorvastatin. Conclusions OxLDL-induced BMP-2 mNRA expression can be suppressed by atorvastatin and probucol, which may be accomplished by activating NF-κB65 expression and upregulating the expression of PPARγ. Our findings also indicate that that BMP-2 mNRA expression includes the activation of reactive oxygen species.

Therapeutic effects of human umbilical cord-derived mesenchymal stem cells against acute tubular necrosis quantified through measures of iNOS, BMP-7 and Bcl-2

Introduction: Acute tubular necrosis (ATN) is the most prevalent cause of acute renal failure (ARF). Mesenchymal stem cell transplantation has been studied as a potential treatment for renal dysfunction due to ATN. Inducible nitric oxide synthase (iNOS), bone morphogenetic protein-7 (BMP-7) and B-cell lymphoma 2 (Bcl-2) are surrogate markers of renal tubular epithelial regeneration and subsequent recovery of renal function following ATN. Methods: Serum creatinine (Scr) and blood urea nitrogen (BUN), as well as expression of iNOS, BMP-7 and Bcl-2 in gentamycin-induced ATN rat kidneys was investigated after human umbilical cord-derived mesenchymal stem cell (HUC-MSC) transplantation. Immunohistochemical staining was performed in 3 groups of rats: gentamycin-induced ATN treated with HUC-MSC, gentamycin-induced ATN without HUC-MSC, and untreated rats not receiving any treatments. Results: HUC-MSC transplantation led to a reduction in Scr and BUN in the kidneys of rats with gentamycin-induced ATN. Expression of iNOS in the HUC-MSC treated group occurred later and the expression levels were much lower during gentamycin-induced ATN compared to rats with ATN that were not treated with HUC-MSC. The expression of BMP-7 and Bcl-2 in the MSC-transplanted group was significantly increased compared to both control groups of rats with injured and healthy renal tubules. Conclusions: HUC-MSCs induce renal protection in a rat model of gentamycin-induced ATN, which is associated with reduced iNOS expression and up-regulation of Bcl-2 and BMP-7.

A Preliminary Mechanism Study on the Treatment of Rabbit Bone Defect Bone Nonunion with Xuduan Jiegu Decoction

Objective: To investigate the curative effect and mechanism of Xuduan Jiegu Decoction in the treatment of rabbit bone defect bone nonunion. Methods: The experiment was randomly divided into three groups;1) The control group that was given neither the modeling nor the treatment;2) The model group that was not given the treatment after the operation to model a bone defect;3) The treatment group that was given intermittent bone decoction after modeling (i.e., the operation), the dose was 105 g/piece, twice a day, for 4 consecutive weeks. For the three groups, Anterolateral X-rays of the left forearm were taken 14 days after the surgery to observe the bone nonunion healing with pretherapy and post-treatment. The expression levels of TGF-β, BMP-2 and VEGF in the blood of each group were measured by ELISA at 4 weeks after treatment. The peri-fracture histopathological changes between each group of pretherapy and post-treatment were evaluated by using tissue sections. Results: Compared with the control group, there was no obvious healing in the model group on the 14th day after the operation. Compared with the model group, the treatment group was treated with Xuduan Jiegu Decoction, and there was a trend of healing and callus formation on the 14th day. HE staining showed that the cells in the control group were closely arranged without any pathological changes. In the model group, the tissues around the fracture end were arranged loosely, and the cells were vacuolated and infiltrated by inflammatory cells. Compared with the model group, the peripheral cell arrangement was better and the peripheral lesions were reduced in the treatment group. The content of TGF-β, BMP-2 and VEGF in blood detected by ELISA was significantly higher in the treatment group than in the control group and the model group, with statistical significance. Conclusion: Xuduan Jiegu Decoction can promote callus formation and accelerate fracture healing in rabbit radius bone defect, and the mechanism of promoting fracture healing is related to the increases of TGF-β, BMP-2 and VEGF levels.

Pilose antler aqueous extract promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by stimulating the BMP-2/Smad1, 5/Runx2 signaling pathway

Peptides from Pilose antler aqueous extract(PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells(BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE’s effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction(Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase(ALP), runt-related transcription factor 2(Runx2), osteocalcin(OCN), bone morphogenetic protein-2(BMP-2), and collagen I(COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200μg·mL^-1 showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.

Mesoporous silica rods with cone shaped pores modulate inflammation and deliver BMP-2 for bone regeneration

Biomaterials with suitable osteoimmunomodulation properties and ability to deliver osteoinductive biomolecules,such as bone morphogenetic proteins,are desired for bone regeneration.Herein,we report the development of mesoporous silica rods with large cone-shaped pores(MSR-CP)to load and deliver large protein drugs.It is noted that those cone-shaped pores on the surface modulated the immune response and reduced the pro-inflammatory reaction of stimulated macrophage.Furthermore,bone morphogenetic proteins 2(BMP-2)loaded MSR-CP facilitated osteogenic differentiation and promoted osteogenesis of bone marrow stromal cells.In vivo tests confirmed BMP-2 loaded MSR-CP improved the bone regeneration performance.This study provides a potential strategy for the design of drug delivery systems for bone regeneration.

Enhancement of BMP-2 and VEGF carried by mineralized collagen for mandibular bone regeneration

Repairing damage in the craniofacial skeleton is challenging.Craniofacial bones require intramembranous ossification to generate tissue-engineered bone grafts via angiogenesis and osteogenesis.Here,we designed a mineralized collagen delivery system for BMP-2 and vascular endothelial growth factor(VEGF)for implantation into animal models of mandibular defects.BMP-2/VEGF were mixed with mineralized collagen which was implanted into the rabbit mandibular.Animals were divided into(i)controls with no growth factors;(ii)BMP-2 alone;or(iii)BMP-2 and VEGF combined.CT and hisomputed tomography and histological staining were performed to assess bone repair.New bone formation was higher in BMP-2 and BMP-2-VEGF groups in which angiogenesis and osteogenesis were enhanced.This highlights the use of mineralized collagen with BMP-2/VEGF as an effective alternative for bone regeneration.

Fabrication and evaluation of a BMP-2/dexamethasone co-loaded gelatin sponge scaffold for rapid bone regeneration

Improving the osteogenic activity of BMP-2 in vivo has significant clinical application value.In this research,we use a clinical gelatin sponge scaffold loaded with BMP-2 and dexamethasone(Dex)to evaluate the osteogenic activity of dual drugs via ectopic osteogenesis in vivo.We also investigate the mechanism of osteogenesis induced by BMP-2 and Dex with C2C12,a multipotent muscle-derived progenitor cell.The results show that the gelatin scaffold with Dex and BMP-2 can significantly accelerate osteogenesis in vivo.It is indicated that compared with the BMP-2 or Dex alone,100nM of Dex can dramatically enhance the BMP-2-induced alkaline phosphatase activity(ALP),ALP mRNA expression and mineralization.Further studies show that 100nM of Dex can maintain the secondary structure of BMP-2 and facilitate recognition of BMP-2 with its receptors on the surface of C2C12 cells.We also find that in C2C12,Dex has no obvious effect on the BMP-2-induced Smad1/5/8 protein expression and the STAT3-dependent pathway,but Runx2-dependent pathway is involved in the Dex-stimulated osteoblast differentiation of BMP-2 both in vitro and in vivo.Based on these results,a potential mechanism model about the synergistic osteoinductive effect of Dex and BMP-2 in C2C12 cells via Runx2 activation is proposed.This may provide a theoretical basis for the pre-clinical application of Dex and BMP-2 for bone regeneration.

Implantable blood clot loaded with BMP-2 for regulation of osteoimmunology and enhancement of bone repair

The treatment of large-area bone defects still faces many difficulties and challenges.Here,we developed a blood clot delivery platform loaded with BMP-2 protein(BMP-2@BC)for enhanced bone regeneration.Blood clot gel platform as natural biomaterials can be engineered from autologous blood.Once implanted into the large bone defect site,it can be used for BMP-2 local delivery,as well as modulating osteoimmunology by recruiting a great number of macrophages and regulating their polarization at different stages.Moreover,due to the deep-red color of blood clot gel,mild localized hyperthermia under laser irradiation further accelerated bone repair and regeneration.We find that the immune niche within the bone defect microenvironment can be modulated in a controllable manner by the blood clots implantation and laser treatment.We further demonstrate that the newly formed bone covered almost 95%of the skull defect area by our strategy in both mice and rat disease models.Due to the great biocompatibility,photothermal potential,and osteoimmunomodulation capacity,such technology shows great promise to be used in further clinical translation.

In vitro and in vivo evaluation of bone morphogenetic protein- 2 （BMP-2） immobilized collagen-coated polyetheretherketone （PEEK）

Polyetheretherketone （PEEK） is regarded as one of the most potential candidates of biomaterials in spinal implant applications. However, as a bioinert material, PEEK plays a limited role in osteoconduction and osseointegration. In this study, recombinant human bone morphogenetic protein-2 （rhBMP-2） was immobilized onto the surface of collagen-coated PEEK in order to prepare a multi-functional material. After adsorbed onto the PEEK surface by hydroph（bic interaction, collagen was cross-linked with N-（3-dimethylaminopropyl）-N＇-ethyl carbodiimide hydrochloride （EDC） and N-hydro xysuccinimide （NHS）. EDCINHS system also contributed to the immobilization of rhBMP- 2. Water contact angle tests, XPS and SEM clearly demonstrated the surface changes. ELISA tests quantified the amount of rhBMP-2 immobilized and the release over a period of 30 d. In vitro evaluation proved that the osteogenesis differentiation rate was higher when cells were cultured on modified PEEK discs than on regular ones. In vivo tests were conducted and positive changes of major parameters were presented. This report demonstrates that the rhBMP-2 immobilized method for PEEK modification increase bioactivity in vitro and in vivo, suggesting its practicability in orthopedic and spinal clinical applications.

Construction of a eukaryotic expression vector pEGFP-C1-BMP-2 and its effect on cell migration

Objective: Bone morphogenetic proteins (BMPs) are known to play an important role in bone and cartilage development. Recent research has shown that BMPs may induce tumorigenesis and promote tumor to spread, but the molecular mechanisms have not been elucidated. The aim of the present study was to investigate the regulatory function of BMP-2 in the migration of COS-7 cells and the underlying mechanisms. Methods: Human BMP-2 genetic fragment was amplified and introduced into the pEGFP-C1 vector. After being confirmed by XhoI and BamHI digestion analyses and DNA sequencing, the recombinant pEGFP-C1-BMP-2 plasmid was transfected into COS-7 cells. The influence of BMP-2 on cell migration and cofilin activity was detected by cell scratch assay and Western blotting. Results: The recombinant pEGFP-C1-BMP-2 was effectively expressed in COS-7 cells. An increased phosphorylation of both LIMK1 and cofilin and an enhancement of cell migration were observed in cells with overexpression of BMP-2. Conclusions: A recombinant pEGFP-C1-BMP-2 vector was successfully constructed and overexpression of BMP-2 regulated the activities of the downstream molecules of the Rho GTPase signaling pathway, which might contribute to the enhancement of cell migration.

骨形态发生蛋白-2基因突变与儿童燃煤型氟骨症的关联

目的 探讨贵州省织金县燃煤型氟中毒病区儿童氟骨症与骨形态发生蛋白-2（bonemorphogeneticproteins．2，BMP-2）基因突变的关联。方法2010年，在贵州省织金县燃煤型氟中毒病区儿童氟骨症情况调查中，通过《氟骨症X线诊断》（WS192．1999）确诊了氟骨症患儿121例，从中选取50例作为氟骨症组；选取该地区经相同诊断方法诊断为无氟中毒骨病变的，且经X线拍片排除佝偻病等相关骨骼疾病的健康儿童30例作为对照组。采用聚合酶链式反应结合DNA测序技术，对氟骨症组及对照组儿童进行BMP．2基因全部3个外显子序列筛查，检测BMP-2基因突变情况。结果①外显子1的第401—402bp处T碱基插入突变：氟骨症组和对照组儿童T碱基插入突变基因型频率分布分别为27．7％（13／47）和7．1％（2／28），两组比较差异有统计学意义（X2=4．600，P〈0．05），调整衄值为4．62（1．94～10．90）。②外显子2的894bp处T-G突变：氟骨症组和对照组儿童TG突变基因型频率分布为14．O％（7／50）和16．7％（5／30），两组比较差异无统计学意义（x2=0．103，P〉0．05）。③外显子2的1046bp处A—G的突变：氟骨症组和对照组基因型分布AA、AG、GG分别为30．0％（15／50）、24．0％（12／50）、46．O％（23／50）和50．0％（15／30）、20．0％（6／30）、30．0％（9／30），组间比较差异无统计学意义（x2=3．099，P〉0．05）。结论外显子1的第401—402bp处T碱基插入突变与氟骨症的关系较为密切，外显子2的1046bp处A—G突变和氟骨症的关系不大。

Dual-response of multi-functional microsphere system to ultrasound and microenvironment for enhanced bone defect treatment

Using bone tissue engineering strategies to achieve bone defect repair is a promising modality.However,the repair process outcomes are often unsatisfactory.Here we properly designed a multi-functional microsphere system,which could deliver bioactive proteins under the dual response of ultrasound and microenvironment,release microenvironment-responsive products on demand,reverse bone injury microenvironment,regulate the immune microenvironment,and achieve excellent bone defect treatment outcomes.In particular,the MnO_(2) introduced into the poly(lactic-co-glycolic acid)(PLGA)microspheres during synthesis could consume the acid produced by the degradation of PLGA to protect bone morphogenetic protein-2(BMP-2).More importantly,MnO_(2) could consume reactive oxygen species(ROS)and produce Mn^(2+)and oxygen(O_(2)),further promoting the repair of bone defects while reversing the microenvironment.Moreover,the reversal of the bone injury microenvironment and the depletion of ROS promoted the polarization of M1 macrophages to M2 macrophages,and the immune microenvironment was regulated.Notably,the ultrasound(US)irradiation used during treatment also allowed the on-demand release of microenvironment-responsive products.The multi-functional microsphere system combines the effects of on-demand delivery,reversal of bone injury microenvironment,and regulation of the immune microenvironment,providing new horizons for the clinical application of protein delivery and bone defect repair.

Recapitulation of growth factor-enriched microenvironment via BMP receptor activating hydrogel

Exposure to a growth factor abundant milieu has remarkable regenerative and rejuvenating effects on organ diseases,tissue damage,and regeneration,including skeletal system defects and bone regeneration.Although the introduction of candidate growth factors into relevant fields has been reported,their regenerative effects remain unsatisfactory,mainly because of the experimental challenges with limited types of growth factors,elusive dosage adjustment,and asynchronous stem cell activation with cytokine secretion.Here,an innovative hydrogel recapitulating a growth factor-enriched microenvironment(GEM)for regenerative advantage,is reported.This sulfated hydrogel includes bone morphogenetic protein-2(BMP-2),an essential growth factor in osteogenesis,to direct mesenchymal stem cell(MSC)differentiation,stimulate cell proliferation,and improve bone formation.The semi-synthetic hydrogel,sulfonated gelatin(S-Gelatin),can amplify BMP-2 signaling in mouse MSCs by enhancing the binding between BMP-2 and BMP-2 type II receptors(BMPR2),which are located on MSC nuclei and activated by the hydrogel.Importantly,the dramatically improved cytokine secretion of MSCs throughout regeneration confirms the growth factor-acquiring potential of S-Gelatin/rhBMP-2 hydrogel,leading to the vascularization enhancement.These findings provide a new strategy to achieve an in situ GEM and accelerated bone regeneration by amplifying the regenerative capacity of rhBMP-2 and capturing endogenous growth factors.