Preparation and osteoinduction of active micro-arc oxidation films on Ti-3Zr-2Sn-3Mo-25Nb alloy

A layer of porous film containing Ca and P was prepared by the micro-arc oxidation method on the surface of a novel near β biomedical Ti-3Zr-2Sn-3Mo-25Nb alloy, and then NH2- active group was introduced to the films by activation treatment. The phase composition, surface micro-topography and elemental characteristics of the micro-arc oxidation films were investigated with XRD, SEM, EDS and XPS, and the osteoinduction of the micro-arc oxidation films was tested using the simulated body fluid immersion, the in-vitro osteoblast cultivation test and animal experiment. The results show that the oxide layer is a kind of porous ceramic intermixture and contains Ca and P. The films in the simulated body fluid can induce apatite formation, resulting in excellent bioactivity. The cell test discovers that osteoblasts can grow well on the surface of micro-arc oxidation films. And the Ti-3Zr-2Sn-3Mo-25Nb biomedical alloy coated with active porous calcium-phosphate films shows better osteoinduction in vivo.

The Role of Microstructure of Highly Purified Beta-Tricalcium Phosphate for Osteoinduction in Canine Dorsal Muscles

Porous β-tricalcium phosphate (TCP) displays osteoinductivity in certain animals in the absence of osteoinductive agents. We evaluated whether the microstructure may be an important determinant of osteoinduction, and also investigated how bone formation was promoted using β-TCP combined with bone marrow aspirates. We prepared two types of β-TCP, namely, β-TCP A, which possessed interconnected macropores and micropores, and β-TCP B, which possessed macropores but had less detectable micropores. These were implanted with or without marrow in canine muscles. Bone formation and the resorption of each β-TCP implant were evaluated histologically. Newly formed bone began to appear at day 42 in the implants of β-TCP A alone, but the implants of β-TCP B alone did not show any bone formation by day 42. Meanwhile, bone formation was already evident on day 14 by loading with bone marrow aspirates with or without micropores. By immunohistochemistry, the number of cathepsin K-positive cells (osteoclasts) increased as time passed in the implants of β-TCP A alone, while the number of the osteoclasts did not change obviously in the implants of β-TCP B alone from day 14 to 56. Reticular fibrils were evident within the β-TCP A, and were barely observed in the β-TCP B in the silver impregnation. The present result would bring about the possible role to enhance the importance of the surface microstructure for the better osteoinductivity. Our findings suggest that the combination of porous β-TCP and bone marrow facilitates bone formation.

Osteoinduction and Osteoconduction with Porous Beta-Tricalcium Phosphate Implanted after Fibular Resection in Humans

Osteoinductive properties of β-TCP remain unknown in humans. It is important to improve the bone grafts which have been the standard treatment for bone defect due to their biocompatibility and bone-healing properties. The purpose of this study was to radiologically clarify the bone forming property of β-TCP by evaluating the replacement of β-TCP by newly formed bone in the defect after fibular resection and to examine the histological features of a β-TCP specimen three months after grafting. Radiographs of 17 patients who underwent β-TCP grafting were evaluated. Osteoinductive and osteoconductive properties were assessed by examining bone formation from the remnant fibula, periosteum, and β-TCP alone. In one case, β-TCP was removed later because of postoperative complications and was evaluated histologically. Twenty two of 34 sites between the remnant fibula and β-TCP had achieved good bone regeneration. Five of 14 sites between the periosteum and β-TCP had achieved good bone regeneration. We found immature but evident bone formation in three cases with no osseous and periosteal sites. Histological analysis revealed bone formation on the outer macropore surface of β-TCP. Some blood vessels formed in the macropores expressed CD31 and CD34, while a few lymphatic vessels expressed CD34 and podoplanin. Thus, the osteoinductive ability of β-TCP alone was demonstrated in humans radiographically for the first time. The histological morphology of β-TCP was demonstrated at an early stage after grafting in humans.

Strategies for creating living, additively manufactured, open-cellular metal and alloy implants by promoting osseointegration,osteoinduction and vascularization: An overview

Additive manufacturing of porous, open-cellular metal or alloy implants, fabricated by laser or electron beam melting of a powder bed, is briefly reviewed in relation to optimizing biomechanical compatibility by assuring elastic(Young's) modulus matching of proximate bone, along with corresponding pore sizes assuring osseointegration and vasculature development and migration. In addition, associated, requisite compressive and fatigue strengths for such implants are described. Strategies for optimizing osteoblast(bone cell) development and osteoinduction as well as vascularization of tissue in 3 D scaffolds and tissue engineering constructs for bone repair are reviewed in relation to the biology of osteogenesis and neovascularization in bone, and the role of associated growth factors, bone morphogenic proteins, signaling molecules and the like. Prospects for infusing hydrogel/collagen matrices containing these cellular and protein components or surgically extracted intramedullary(bone marrow) concentrate/aspirate containing these biological and cell components into porous implants are discussed, as strategies for creating living implants, which over the long term would act as metal or alloy scaffolds.

Osteoinduction by Ca-P biomaterials implanted into the muscles of mice

The osteoinduction of porous biphasic calcium phosphate ceramics (BCP) has been widely reported and documented,but little research has been performed on rodent animals,e.g.,mice.In this study,we report osteoinduction in a mouse model.Thirty mice were divided into two groups.BCP materials (Sample A) and control ceramics (Sample B) were implanted into the leg muscle,respectively.Five mice in each group were killed at 15,30,and 45 d after surgery.Sample A and Sample B were harvested and used for hematoxylin and eosin (HE) staining,immunohistochemistry (IHC) staining,and Alizarin Red S staining to check bone formation in the biomaterials.Histological analysis showed that no bone tissue was formed 15 d after implantation (0/5) in either of the two groups.Newly-formed bone tissues were observed in Sample A at 30 d (5/5) and 45 d (5/5) after implantation;the average amounts of newly-formed bone tissues were approximately 5.2% and 8.6%,respectively.However,we did not see any bone tissue in Sample B until 45 d after implantation.Bone-related molecular makers such as bone morphogenesis protein-2 (BMP-2),collagen type I,and osteopontin were detected by IHC staining in Sample A 30 d after implantation.In addition,the newly-formed bone was also confirmed by Alizarin Red S staining.Because this is the report of osteoinduction in the rodent animal on which all the biotechnologies were available,our results may contribute to further mechanism research.

Preparation of multigradient hydroxyapatite scaffolds and evaluation of their osteoinduction properties

Porous hydroxyapatite(HA)scaffolds are often used as bone repair materials,owing to their good biocompatibility,osteoconductivity and low cost.Vascularization and osteoinductivity of porous HA scaffolds were limited in clinical application,and these disadvantages were need to be improved urgently.We used water-in-oil gelation and pore former methods to prepare HA spheres and a porous cylindrical HA container,respectively.The prepared HA spheres were filled in container to assemble into composite scaffold.By adjusting the solid content of the slurry(solid mixture of chitin sol and HA powder)and the sintering temperature,the porosity and crystallinity of the HA spheres could be significantly improved;and mineralization of the HA spheres significantly improved the biological activity of the composite scaffold.The multigradient(porosity,crystallinity and mineralization)scaffold(HA-700)filled with the mineralized HA spheres exhibited a lower compressive strength;however,in vivo results showed that their vascularization ability were higher than those of other groups,and their osteogenic Gini index(Go:an index of bone mass,and inversely proportional to bone mass)showed a continuous decrease with the implantation time.This study provides a new method to improve porous HA scaffolds and meet the demands of bone tissue engineering applications.

Design and evaluation of a novel sub-scaffold dental implant system based on the osteoinduction of micro-nano bioactive glass

Alveolar ridge atrophy brings great challenges for endosteal implantation due to the lack of adequate vertical bone mass to hold the implants.To overcome this limitation,we developed a novel dental implant design:sub-scaffold dental implant system(SDIS),which is composed of a metal implant and a micro-nano bioactive glass scaffold.This implant system can be directly implanted under mucous membranes without adding any biomolecules or destroying the alveolar ridge.To evaluate the performance of the novel implant system in vivo,SDISs were implanted into the subepicranial aponeurosis space of Sprague–Dawley rats.After 6 weeks,the SDIS and surrounding tissues were collected and analysed by micro-CT,scanning electron microscopy and histology.Our results showed that SDISs implanted into the sub-epicranial aponeurosis had integrated with the skull without any mobility and could stably support a denture.Moreover,this design achieved alveolar ridge augmentation,as active osteogenesis could be observed outside the cortical bone.Considering that the microenvironment of the sub-epicranial aponeurosis space is similar to that of the alveolar ridge,SDISs have great potential for clinical applications in the treatment of atrophic alveolar ridges.The study was approved by the Animal Care Committee of Guangdong Pharmaceutical University(approval No.2017370).

A fast on-demand preparation of injectable self-healing nanocomposite hydrogels for efficient osteoinduction

Injectable hydrogels have been considered as promising materials for bone regeneration,but their osteoinduction and mechanical performance are yet to be improved.In this study,a novel biocompatible injectable and self-healing nano hybrid hydrogel was on-demand prepared via a fast(within 30 s)and easy gelation approach by reversible Schiff base formed between-CH=O of oxidized sodium alginate(OSA)and-NH2 of glycol chitosan(GCS)mixed with calcium phosphate nanoparticles(CaP NPs).Its raw materials can be ready in large quantities by a simple synthesis process.The mechanical strength,degradation and swelling behavior of the hydrogel can be readily controlled by simply controlling the molar ratio of-CH=O and-NH2.This hydrogel exhibits pH responsiveness,good degradability and biocompatibility.The hydrogel used as the matrix for mesenchymal stem cells can significantly induce the proliferation,differentiation and osteoinduction in vitro.These results showed this novel hydrogel is an ideal candidate for applications in bone tissue regeneration and drug delivery.

Ectopic Bone Formation in vivo Induced by a Novel Synthetic Peptide Derived from BMP-2 Using Porous Collagen Scaffolds

To investigate the osteoinductive and ectopicly osteogenic effects of a novel peptide P24 derived from bone morphogenetic protein 2 （BMP2）, biodegradable collagen scaffolds （CS） were used to load BMP-2-derived peptide solutions with different concentrations （0.4 mg peptide/CS, 0.1 mg peptide/CS and pure CS, respectively）, and the implants were implanted into muscular pockets on the back of Wistar rats. Radiographs and histological analysis were performed to evaluate the ectopic bone effects. Active ectopic bone formation was seen in both groups containing the peptide at different concentration （0.4 mg and 0.1 mg）, whereas no bone formation and only fibrous tissue was seen in the pure CS group. The new bone formation induced by the peptide P24 displayed a dose-dependent and time-dependent efficiency. The new bone formation in the 0.4 mg peptide/CS group significantly increased than that of the 0.1 mg peptide/CS group. This novel BMP-2-derived peptide had excellent osteoinductive and ectopicly osteogenic properties which were similar to those of BMP2.

Porous Calcium Phosphate Ceramic Scaffolds for Tissue Engineering

This study examined the biological response of two porous calcium phosphate ceramics, hydroxyapntite （ HA ） and hydroxyapaptite/β-tricalcium phosphate （ HA/β- TCP ） scaffolds. Three different cell types , a human osteoblastic cell line （ HOS ） , primary human osteoblasts （HOB） and human mesenehymal stem cells （MSCs）, were used to examine biocompatibility and osteoinductive capacity. The experimental results showed both materials were highly biocompatible and proliferation was significantly greater on pure HA （ P 〈 0.01 ）, with a peak in proliferation at day 7. Protein levels were significantly higher （ P 〈 0.05） than the control Thermanox（ TMX （ tm） ） for both test materials. Osteoinduction of MSCs was observed on both test materials, with cells seeded on HA/ β-TCP showing greater alkaline phosphatase activity compared to HA alone, indicating an enhancement in osteoinductive property. Both materials show good potential for use as tissue engineered scaffolds.

In Vitro Characterizations of PLLA/β-TCP Porous Matrix Materials and RMSC-PLLA-β-TCP Composite Scaffolds

To develop a novel degradable poly (L-lactic acid)/β-tricalcium phosphate (PLLA/β-TCP) bioactive materials for bone tissueengineering, β-TCP powder was produced by a new wet process. Porous scaffolds were prepared by three steps, i.e. solventcasting, compression molding and leaching stage. Factors influencing the compressive strength and the degradation behaviorof the porous scaffold, e.g. weight fraction of pore forming agent-sodium chloride (NaCl), weight ratio of PLLA: β-TCP,the particle size of β-TCP and the porosity, were discussed in details. Rat marrow stromal cells (RMSC) were incorporatedinto the composite by tissue engineering approach. Biological and osteogenesis potential of the composite scaffold weredetermined with MTT assay, alkaline phosphatase (ALP) activity and bone osteocalcin (OCN) content evaluation. Resultsshow that PLLA/β-TCP bioactive porous scaffold has good mechanical and pore structure with adjustable compressive strengthneeded for surgery. RMSCs seeding on porous PLLA/β-TCP composite behaves good seeding efficacy, biocompatibility andosteoinductive potential. Osteoprogenitor cells could well penetrate into the material matrix and begin cell proliferation andosteogenic differentiation. Osseous matrix could be formed on the surface of the composite after culturing in vitro. It isexpected that the PLLA/β-TCP porous composites are promising scaffolds for bone tissue engineering in prosthesis surgery.

Heterotopic ossification after the use of recombinant human bone morphogenetic protein-7

AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7(rhB MP-7) for the treatment of nonunions.METHODS Bone morphogenetic proteins(BMPs) promote bone formation by auto-induction. Recombinant human BMP-7 in combination with bone grafts was used in 84 patients for the treatment of long bone nonunions. All patients were evaluated radiographicaly for the development of heterotopic ossification during the standard assessment for the nonunion healing. In all patients(80.9%) with radiographic signs of heterotopic ossification, a CT scan was performed. Nonunion site palpation and ROM evaluation of the adjacent jointswere also carried out. Factors related to the patient(age, gender), the nonunion(location, size, chronicity, number of previous procedures, infection, surrounding tissues condition) and the surgical procedure(graft and fixation type, amount of rhB MP-7) were correlated with the development of heterotopic ossification and statistical analysis with Pearsons χ~2 test was performed.RESULTS Eighty point nine percent of the nonunions treated with rh BMP-7, healed with no need for further procedures. Heterotopic bone formation occurred in 15 of 84 patients(17.8%) and it was apparent in the routine radiologi-cal evaluation of the nonunion site, in a mean time of 5.5 mo after the rh BMP-7 application(range 3-12). The heterotopic ossification was located at the femur in 8 cases, at the tibia in 6, and at the humerus in οne patient. In 4 patients a palpable mass was present and only in one patient, with a para-articular knee nonunion treated with rhB MP-7, the size of heterotopic ossification affected the knee range of motion. All the patients with heterotopic ossification were male. Statistical analysis proved that patient's gender was the only important factor for the development of heterotopic ossification(P = 0.007). CONCLUSION Heterotopic ossification after the use of rh BMP-7 in nonunions was common but it did not compromise the final clinical outcome in most cases, and affected only male patients.

Immobilization of RGD Peptide onto the Surface of Apatite-Wollastonite Ceramic for Enhanced Osteoblast Adhesion and Bone Regeneration

The arginine-glycine-aspartic （RGD） acid peptide was grafted to the surface of apatitewollastonite （AW） ceramic in an effort to improve its cell adhesion, proliferation and osteoinduction. RGD peptide was covalently immobilized onto the surface of AW ceramic via the synthetic cross linker AA.PTS-E and 1-ethyl-3-（3-dimethylaminopropyl）carbodiimide （EDC）. The modified surfaces were characterized by attenuated total reflectance Fourier transform infrared spectroscopy （ATR-FTIR） and X-ray photoelectron spectroscopy （XPS）. The chemical analysis indicated that RGD peptide had been immobilized onto the AW surface successfully. The growth of osteoblast-like cells （MG63） showed that modifying the AW surface with RGD peptide enhanced the cell adhesion and proliferation. And the histological evaluation of RGD-AW showed that the bone regeneration and remodeling process were significantly enhanced compared to the original AW ceramics after 2, 4 and 8 weeks implantation in rabbit＇s femoral condyles.

Tissue Response of an Osteoinductive Bioceramic in Bone Defect Rabbit Model

HA/TCP and HA rods （φ5 mm×10 mm） were made for implantation in New Zealand white rabbit with different condition. Sixty three rabbit were divided into three groups： group 1 （n=18）, group 2 （n=27） and group 3 （n=18）. In group 1, 10 mm radius was defected, and one HA/TCP rod was implanted in the muscle a distant away from the bone defect area. In group 2, also, 10 mm radius was defected, one HA rod was implanted in the muscle a distant away from the bone defect area. In group 3, two HA/TCP rods were implanted in the dorsal muscle of the rabbit with bone intact. Histological observation showed that in group 1, some new bone was found only two months after implantation （n=2）, and obvious immature woven bone could be observed in these bioceramics from the 3rd month on. However, in group 3, bone began to be found 6 months after implantation （n=2）. In group 2, we could not find any bone tissue up to 9 month’s observation. These results suggest that, first, the bone defect model could significantly accelerate bone formation at non-osseous sites in rabbits; second,. HA/TCP bioceramics were confirmed with osteoinductive property while HA bioceramics without osteoinductive property nearly. Thus, bone defect might be a good animal model for further researches for osteoinductive bioceramics.

The Clinical Application of Human Bone Matrix Gelatin

This paper reports the results of 24 cases of bone defect resulting from bone tumor or tumor condition excision, and of posterior spinal fusion, treated by human bone matrix gelatin. The success rate of bone defect repair and spinal fusion is 91. 67 %. The results suggest that human bone matrix gelatin has. excellent osteoinductive effect and is ideal substitute for bone autografts.

Induced Bone by Calcium Ohosphate (Ca-P) Ceramics after 2-years Implantation

The changes of induced bone with four kinds of Ca-P ceramics after 2 years implantation in the dorsal muscles of rabbits were investigated. After 2 years implantation, mature bone （with a lamellar structure after the remodeling process） with bone marrow was observed in HA1100 （HA, micro＋macro porous）, HA900 （HA, micro＋macro porous） and BCP （HA/TCP=7：3, micro＋macro porous）, and no bone formation was observed in HA1200 （HA, macro porous） ceramics. The induced bone neither disappeared nor grew uncontrollably, and was seen inside the pores of the implants or on the outer surface under the fibrous layer after 2 years. No bone formation was observed in the soft tissues distant from the implants. These results indicate that heterotopic bone formation induced by Ca-P materials did not give rise to uncontrolled growth over time, and the induced bone is limited to the inside or around the implants. Moreover, ectopic bone formation on the outer surface of the implants was detected in rabbits after 2 years.

Dual-Wavelength Photosensitive Nano-in-Micro Scaffold Regulates Innate and Adaptive Immune Responses for Osteogenesis

The immune response of a biomaterial determines its osteoinductive effect.Although the mechanisms by which some immune cells promote regeneration have been revealed,the biomaterial-induced immune response is a dynamic process involving multiple cells.Currently,it is challenging to accurately regulate the innate and adaptive immune responses to promote osteoinduction in biomaterials.Herein,we investigated the roles of macrophages and dendritic cells(DCs)during the osteoinduction of biphasic calcium phosphate(BCP)scaffolds.We found that osteoinductive BCP directed M2 macrophage polarization and inhibited DC maturation,resulting in low T cell response and efficient osteogenesis.Accordingly,a dual-targeting nano-in-micro scaffold(BCP loaded with gold nanocage,BCP-GNC)was designed to regulate the immune responses of macrophages and DCs.Through a dual-wavelength photosensitive switch,BCP-GNC releases interleukin-4 in the early stage of osteoinduction to target M2 macrophages and then releases dexamethasone in the later stage to target immature DCs,creating a desirable inflammatory environment for osteogenesis.This study demonstrates that biomaterials developed to have specific regulatory capacities for immune cells can be used to control the early inflammatory responses of implanted materials and induce osteogenesis.

Experimental Study of Porous Beta-tricalcium Phosphate and Bone Morphogenetic Protein/porous Beta-tricalcium Phosphate Complex in Rabbit Mandibular Reconstruction

We investigated the osteogenic characteristic and biodegradation behavior of porous beta-tricalcitum phosphate （ β- TCP ） and bone morphogenetic protein/beta-tricalcium phosphate （ BMP/ β- TCP ） complex in mandibular reconstruction and evaluated the advantages of BMP / β- TCP complex in repairing bone defects. Bone defects created in the lower margin of bilateral mandible bodies in 12 rabbits were repaired with β- TCP （ control group ） and BMP/ β- TCP complex （ experimental group ） respectively. The rabbits were euthanized after 2, 4, 8 and 12 weeks and examined by macroscopy, radiography, histology, histomorphometry and image analysis. 2 weeks after surgery, ossftcation of newly-generated tissue in BMP/ β- TCP complex group appeared and after 12 weeks, massive new bone and ossification maturation were seen. However, in β- TCP group without BMP , ossification was not found until 12 weeks after operation. Image analysis showed that bone regeneration rate of BMP/ β- TCP was 30%-40% higher and the degradation rate was 20%- 30% higher than that of β- TCP. Therefore, as a reconstructive material for bone defects, BMP/ β-TCP complex is superior to β-TCP and can be used in oral and maxillofacial surgery.

CORAL AS A CARRIER FOR RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2

By combining coral with recombinant human bone morphogenetic protein-2 (rhBMP-2), rhBMP-2/coral composite was obtained in this study. Following implantation of the composite into the muscle pouches of mice, cartilage growth was induced in the pores or on the surface of the implants at one week, woven bone at three week and lamellar bone with bone marrow at six week, and coral was absorbed partially. The induced formation of endochondral bone was time-related and rhBMP-2 dose-related. The results of this study indicate that the composite possesses a superior ability of osteogenesis, and coral acts as one of the most suitable rhBMP-2 slowrelease carriers currently available. The composite will be a new type of bone substitute to be used in orthopaedics and maxillofacial surgery.

Local Application of Alendronate on <i>β</i>-Tricalcium Phosphate Accelerated Induction of Osteogenesis with Formation of Giant Osteoclast-Like Cell

Intrinsic osteoinductivity—the ability to induce bone formation in ectopic sites without addition of osteogenic factors has been reported in various porous materials. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells are thought to play an important role in material-induced osteoinduction. To investigate the influence of osteoclastic activity on intrinsic osteoinduction, we loaded alendronate (10–2 , 10–4 , and 10–6 M) onto porous β-tricalcium phosphate (β-TCP) blocks to inhibit osteoclastic activity, and evaluated osteoinductivity by implantation of the blocks into the dorsal muscles of adult beagle dogs. Alendronate-loaded porous β-TCP blocks increased both speed and amount of osteoinduction, as measured 4 weeks after implantation, with the 10–4 M alendronate-loaded β-TCP being especially active. This finding indicates that β-TCP loaded with 10–4 M alendronate might prove crucial in providing the desirable balance between the degradation rate of bone scaffolds and their osteoinductive replacement. Thus, material-induced osteoinduction may be controlled by local application of alendronate, establishing alendronate loading as a promising therapeutic approach.