Wei Zhi

Promotion of skin regeneration in diabetic rats by electrospun core-sheath fibers loaded with basic fibroblast growth factor

Diabetic skin ulcer is difficult to heal due to the lack of cellular and molecular signals required for normal wound repair. Emulsion electrospinning was adopted to imbed basic fibroblast growth factor (bFGF) into ultrafine fibers with a core-sheath structure to promote the wound healing process. An initially burst release as low as 14.0 ± 2.2% was achieved, followed by gradual release for around 4 wk. In vitro investigations on mouse embryo fibroblasts indicated that bFGF-loaded fibrous mats enhanced cell adhesion, proliferation, and secretion of extracellular matrix (ECM). Skin wounds were created in the dorsal area of diabetic rats for in vivo evaluation of skin regeneration after covered with bFGF-loaded fibrous mats. Compared with fibrous mats infiltrated with free bFGF, bFGF-loaded scaffolds revealed significantly higher wound recovery rate with complete re-epithelialization and regeneration of skin appendages. Higher density and mature capillary vessels were generated during 2 wk after treatment with bFGF-loaded fibers, and there was no fiber fragment observed in the histological sections at week 4 after operation. The gradual release of bFGF from fibrous mats enhanced collagen deposition and ECM remodeling, and the arrangement and component of collagen fibers were similar to normal tissues. The above results demonstrate the potential use of bFGF-loaded electrospun fibrous mats to rapidly restore the structural and functional properties of wounded skin for patients with diabetic mellitus.

Silver Nanoparticles and Growth Factors Incorporated Hydroxyapatite Coatings on Metallic Implant Surfaces for Enhancement of Osteoinductivity and Antibacterial Properties

Research on incorporation of both growth factors and silver (Ag) into hydroxyapatite (HA) coatings on metallic implant surfaces for enhancing osteoinductivity and antibacterial properties is a challenging work. Generally, Ag nanoparticles are easy to agglomerate and lead to a large increase in local Ag concentration, which could potentially affect cell activity. On the other hand, growth factors immobilization requires mild processing conditions so as to maintain their activities. In this study, bone morphology protein-2 (BMP-2) and Ag nanoparticle contained HA coatings were prepared on Ti surfaces by combining electrochemical deposition (ED) of Ag and electrostatic immobilization of BMP-2. During the ED process, chitosan (CS) was selected as the stabilizing agent to chelate Ag ions and generate Ag nanoparticles that are uniformly distributed in the coatings. CS also reduces Ag toxicity while retaining its antibacterial activity. Afterwards, a BMP/heparin solution was absorbed on the CS/Ag/HA coatings. Consequently, BMP-2 was immobilized on the coatings by the electrostatic attraction between CS, heparin, and BMP-2. Sustained release of BMP-2 and Ag ions from HA coatings was successfully demonstrated for a long period. Results of antibacterial tests indicate that the CS/Ag/HA coatings have high antibacterial properties against both Staphylococcus epidermidis and Escherichia coli. Osteoblasts (OB) culture reveals that the CS/Ag/HA coatings exhibit good biocompatibility. Bone marrow stromal cells (BMSCs) culture indicates that the BMP/CS/Ag/HA coatings have good osteoinductivity and promote the differentiation of BMSCs. Ti bars with BMP/CS/Ag/HA coatings were implanted into the femur of rabbits to evaluate the osteoinductivity of the coatings. Results indicate that BMP/CS/Ag/HA coatings favor bone formation in vivo. In summary, this study presents a convenient and effective method for the incorporation of growth factors and antibacterial agents into HA coatings. This method can be utilized to modify a variety of metallic implant surfaces.

BMP‐2 encapsulated polysaccharide nanoparticle modified biphasic calcium phosphate scaffolds for bone tissue regeneration

Bone morphology protein-2 (BMP-2) encapsulated chitosan/chondrotin sulfate nanoparticles (CHI/CS NPs) are developed to enhance ectopic bone formation on biphasic calcium phosphate (BCP) scaffolds. BMP-2 contained CHI/CS NPs were prepared by a simple and mild polyelectrolyte complexation process. It does not involve harsh organic solvents and high temperature, and therefore retain growth factors activity. These NPs were immobilized on BCP scaffolds, and realize the sustained release of growth factors from the scaffolds. The bare BCP scaffolds, NP loaded scaffolds (BCP-NP), and NP loaded and polydopamine coated scaffolds (BCP-Dop-NP) were seeded with bone marrow stroma cells (BMSC) to evaluate the osteoinductivity of the scaffolds. BMSC culture results indicate that all scaffolds favor cell adhesion, proliferation, differentiation. Afterwards, the bare BCP, BCP-NP, and BCP-Dop-NP scaffolds were implanted into rabbits intramuscularly to evaluate the ectopic bone formation of scaffolds. In vivo results indicate that the BCP-NP and BCP-Dop-NP scaffolds enhance more ectopic bone formation than the bare BCP scaffolds. Both the in vitro and in vivo results demonstrate that BMP-2 encapsulated polysaccharide NPs are effective to improve the osteoinductivity of the scaffolds. In addition, BCP-NP scaffolds induce more bone formation than BCP-Dop-NP scaffolds. This is because BCP-NP scaffolds harness the intrinsic osteoinductivity BCP and BMP-2, whereas BCP-Dop-NP scaffolds have polydopamine coatings that inhibit the surfaces biological features of BCP scaffolds, and therefore weaken the bone formation ability of scaffolds.

Fabrication of TiO2 nanotubes on porous titanium scaffold and biocompatibility evaluation in vitro and in vivo

Porous titanium was modified by anodic oxidation and heat treatment method. Scanning electron microscopy and X-ray diffraction examinations revealed that the modified surface of porous titanium was covered by anatase nanotubes. In vitro, the bioactivity of specimens before and after modification was evaluated by immersing into the double-concentration simulated body fluid for 7 days. The porous titanium specimens were implanted into the femurs of dogs for 3 months. The osteointegration of the implants was investigated by push-out test and histological examination. The results showed that the porous titanium with anatase nanotubes has the superior ability of apatite formation and a higher push-out force when compared with the other implants. The histological analysis indicated that the implant with anatase nanotubes had excellent ability to facilitate the osteointegration in vivo.

A novel porous bioceramics scaffold by accumulating hydroxyapatite spherulites for large bone tissue engineering in vivo. II. Construct large volume of bone grafts.

In vivo engineering of bone autografts using bioceramic scaffolds with appropriate porous structures is a potential approach to prepare autologous bone grafts for the repair of critical-sized bone defects. This study investigated the evolutionary process of osteogenesis, angiogenesis, and compressive strength of bioceramic scaffolds implanted in two non-osseous sites of dogs: the abdominal cavity and the dorsal muscle. Hydroxyapatite (HA) sphere-accumulated scaffolds with controlled porous structures were prepared and placed in the two sites for up to 6 months. Analyses of retrieved scaffolds found that osteogenesis and angiogenesis were faster in scaffolds implanted in dorsal muscles compared with those placed in abdominal cavities. The abdominal cavity, however, can accommodate larger bone grafts with designed shape. Analyses of scaffolds implanted in abdominal cavities [an environment of a low mesenchymal stem cell (MSC) density] further demonstrated that angiogenesis play critical roles during osteogenesis in the scaffolds, presumably by supplying progenitor cells and/or MSCs as seed cells. This study also examined the relationship between the volume of bone grafts and the physiological environment of in vivo bioreactor. These results provide basic information for the selection of appropriate implanting sites and culture time required to engineer autologous bone grafts for the clinical bone defect repair. Based on these positive results, a pilot study has applied the grafts constructed in canine abdominal cavity to repair segmental bone defect in load-bearing sites (limbs).

Study of bilineage differentiation of human-bone-marrow-derived mesenchymal stem cells in oxidized sodium alginate/N-succinyl chitosan hydrogels and synergistic effects of RGD modification and low-intensity pulsed ultrasound.

The level of formation of new bone and vascularization in bone tissue engineering scaffold implants is considered as a critical factor for clinical application. In this study, an approach using an RGD-grafted oxidized sodium alginate/N-succinyl chitosan (RGD-OSA/NSC) hydrogel as a scaffold and low-intensity pulsed ultrasound (LIPUS) as mechanical stimulation was proposed to achieve a high level of formation of new bone and vascularization. An in vitro study of endothelial and osteogenic differentiations of human-bone-marrow-derived mesenchymal stem cells (hMSCs) was conducted to evaluate it. The results showed that RGD-OSA/NSC composite hydrogels presented good biological properties in attachment, proliferation and differentiation of cells. The MTT cell viability assay showed that the total number of cells increased more significantly in the LIPUS-stimulated groups with RGD than that in the control ones; similar results were obtained for alkaline phosphatase activity/staining and mineralized nodule formation assay of osteogenic induction and immunohistochemical test of endothelial induction. The positive synergistic effect of LIPUS and RGD on the enhancement of proliferation and differentiation of hMSCs was observed. These findings suggest that the hybrid use of RGD modification and LIPUS might provide one approach to achieve a high level of formation of new bone and vascularization in bone tissue engineering scaffold implants.

Histomorphological researches on large porous hydroxyapatite cylinder tubes with polylactic acid surface coating in different nonskeletal sites in vivo.

Porous hydroxyapatite (HA) ceramic cylinder tubes coated with polylactic acid on the exposed surfaces were implanted in four nonskeletal sites (omentum, peritoneum, vastus lateralis, and side of femur). Six months postoperatively, proper amount of Chinese ink was injected to dye the implanting areas. Decalcified and nondecalcified sections were observed under inverted microscope. The results showed that the soft tissues around the HA cylinder tubes in peritoneum, vastus lateralis, and side of femur groups appeared visible black. Some small blacked vascular architectures were also discernible. However in omentum group, only small number of blacked vessels existed. Histological observations indicated that vascularization and ossification occurred in peritoneum, vastus lateralis, and side of femur groups. In omentum group, there was no any sign of vascularization and ossification. A conclusion could be made in this study that excepting bones and muscles, parietal peritoneum could serve as a potential spot for culturing histoengineering hydroxyapatite (HA)-polylactic acid (PLA) ceramic bone substitutes.

Ectopic osteogenesis and angiogenesis regulated by porous architecture of hydroxyapatite scaffolds with similar interconnecting structure in vivo

The macro-pore sizes of porous scaffold play a key role for regulating ectopic osteogenesis and angiogenesis but many researches ignored the influence of interconnection between macro-pores with different sizes. In order to accurately reveal the relationship between ectopic osteogenesis and macro-pore sizes in dorsal muscle and abdominal cavities of dogs, hydroxyapatite (HA) scaffolds with three different macro-pore sizes of 500–650, 750–900 and 1100–1250 µm were prepared via sugar spheres-leaching process, which also had similar interconnecting structure determined by keeping the d/s ratio of interconnecting window diameter to macro-pore size constant. The permeability test showed that the seepage flow of fluid through the porous scaffolds increased with the increase of macro-pore sizes. The cell growth in three scaffolds was not affected by the macro-pore sizes. The in vivo ectopic implantation results indicated that the macro-pore sizes of HA scaffolds with the similar interconnecting structure have impact not only the speed of osteogenesis and angiogenesis but also the space distribution of newly formed bone. The scaffold with macro-pore sizes of 750–900 µm exhibited much faster angiogenesis and osteogenesis, and much more uniformly distribution of new bone than those with other macro-pore sizes. This work illustrates the importance of a suitable macro-pore sizes in HA scaffolds with the similar interconnecting structure which provides the environment for ectopic osteogenesis and angiogenesis.

Porous nanoapatite scaffolds synthesized using an approach of interfacial mineralization reaction and their bioactivity.

There is a growing interest in the use of calcium phosphate, used to fabricate porous scaffolds for bone tissue regeneration and repair. However, it is difficult to obtain interconnected pores with very high porosity and to engineer the topography of the pore walls for calcium phosphate ceramic scaffolds. In this study, a novelty method interfacial mineralization reaction was used to fabricate porous nano-calcium phosphate ceramic scaffolds with three-dimensional surface topography of walls, which was tuned using different surfactants; using this method, porous scaffolds with different shapes were obtained, which demonstrates that interfacial mineralization reaction is not only a good method to prepare porous ceramic scaffolds of calcium phosphate but also an efficient approach to engineer the topography of the pore walls. The as-prepared porous ceramic scaffolds have also been proved to have good biocompatibility, bioactivity, and biodegradability, which are necessary for the clinical application. In vivo experimental results revealed that not only osteoconduction but also osteoinduction was responsible for the bone formation in our scaffolds, which accelerated the formation of new bone, and that the degradation process of our porous scaffolds could match osteoinduction, mineralization of matrix and bone, and reconstruction of new bone very well, and porous scaffolds could be completely substituted by the new bone.

Molecular docking characterization of a four-domain segment of human fibronectin encompassing the RGD loop with hydroxyapatite.

Fibronectin adsorption on biomaterial surfaces plays a key role in the biocompatibility of biomedical implants. In the current study, the adsorption behavior of the 7–10th type III modules of fibronectin (FN-III7–10) in the presence of hydroxyapatite (HAP) was systematically investigated by using molecular docking approach. It was revealed that the FN-III10 is the most important module among FN-III7–10 in promoting fibronectin binding to HAP by optimizing the interaction energy; the arginine residues were observed to directly interact with the hydroxyl group of HAP through electrostatic forces and hydrogen bonding. Moreover, it was found that the HAP-binding sites on FN-III10 are mainly located at the RGD loop region, which does not affect the interaction between the fibronectin protein and its cognate receptors on the cell surface.