Implants of polyanionic collagen matrix in bone defects of ovariectomized rats

In recent years, there has been a great interest in the development of biomaterials that could be used in the repair of bone defects. Collagen matrix (CM) has the advantage that it can be modified chemically to improve its mechanical properties. The aim of the present study was to evaluate the effect of three-dimensional membranes of native or anionic (submitted to alkaline treatment for 48 or 96 h) collagen matrix on the consolidation of osteoporosis bone fractures resulting from the gonadal hormone alterations caused by ovariectomy in rats subjected to hormone replacement therapy. The animals received the implants 4 months after ovariectomy and were sacrificed 8 weeks after implantation of the membranes into 4-mm wide bone defects created in the distal third of the femur with a surgical bur. Macroscopic analysis revealed the absence of pathological alterations in the implanted areas, suggesting that the material was biocompatible. Microscopic analysis showed a lower amount of bone ingrowth in the areas receiving the native membrane compared to the bone defects filled with the anionic membranes. In ovariectomized animals receiving anionic membranes, a delay in bone regeneration was observed mainly in animals not subjected to hormone replacement therapy. We conclude that anionic membranes treated with alkaline solution for 48 and 96 h presented better results in terms of bone ingrowth.     

Osseous regeneration in the presence of oxidized cellulose and collagen

Oxidized cellulose and collagen are two absorbable hemostatic scaffolding materials that are used widely in surgery. A histomorphological study was undertaken to determine the tissue response and extent of healing brought about by intraosseously implanting these two materials in the femur and tibia of sheep. There was no major difference in the rate of repair of the bone defects brought about by these two materials, with the bone defects being completely repaired by lamellar bone at 6-8 weeks. Therefore, our results suggest that, in most instances where collagen is presently used in surgical applications, it could be substituted by oxidized cellulose.     

Bone regeneration potential of a soybean-based filler: experimental study in a rabbit cancellous bone defects

Autologous and allogenic bone grafts are considered as materials of choice for bone reconstructive surgery, but limited availability, risks of transmittable diseases and inconsistent clinical performances have prompted the development of alternative biomaterials. The present work compares the bone regeneration potential of a soybean based bone filler (SB bone filler) in comparison to a commercial 50:50 poly(d,l lactide–glycolide)-based bone graft (Fisiograft^® gel) when implanted into a critical size defect (6-mm diameter, 10-mm length) in rabbit distal femurs. The histomorphometric and microhardness analyses of femoral condyles 4, 8, 16 and 24 weeks after surgery showed that no significant difference was found in the percentage of both bone repair and bone in-growth in the external, medium and inner defect areas. The SB filler-treated defects showed significantly higher outer bone formation and microhardness results at 24 weeks than Fisiograft^® gel (P < 0.05). Soybean-based biomaterials clearly promoted bone repair through a mechanism of action that is likely to involve both the scaffolding role of the biomaterial for osteoblasts and the induction of their differentiation.     

Preparation of a biphasic scaffold for osteochondral tissue engineering

Tissue engineering has been developed as a prospective approach for the repair of articular cartilage defects. Engineered osteochondral implants can facilitate the fixation and integration with host tissue, and therefore promote the regeneration of osteochondral defects. A biphasic scaffold with a stratified two-layer structure for osteochondral tissue engineering was developed from biodegradable synthetic and naturally derived polymers. The upper layer of the scaffold for cartilage engineering was collagen sponge; the lower layer for bone engineering was a composite sponge of poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen. The PLGA–collagen composite sponge layer had a composite structure with collagen microsponge formed in the pores of a skeleton PLGA sponge. The collagen sponge in the two respective layers was connected. Observation of the collagen/PLGA–collagen biphasic scaffold by scanning electron microscopy (SEM) demonstrated the connected stratified structure. The biphasic scaffold was used for culture of canine bone-marrow-derived mesenchymal stem cells. The cell/scaffold construct was implanted in an osteochondral defect in the knee of a one-year old beagle. Osteochondral tissue was regenerated four months after implantation. Cartilage- and bone-like tissues were formed in the respective layers. The collagen/PLGA–collagen biphasic scaffold will be useful for osteochondral tissue engineering.     

Biocompatibility of calcium phosphate bone cement with optimised mechanical properties: an in vivo study

This work establishes the in vivo performance of modified calcium phosphate bone cements for vertebroplasty of spinal fractures using a lapine model. A non-modified calcium phosphate bone cement and collagen-calcium phosphate bone cements composites with enhanced mechanical properties, utilising either bovine collagen or collagen from a marine sponge, were compared to a commercial poly(methyl methacrylate) cement. Conical cement samples (8?mm height?×?4?mm base diameter) were press-fit into distal femoral condyle defects in New Zealand White rabbits and assessed after 5 and 10 weeks. Bone apposition and tartrate-resistant acid phosphatase activity around cements were assessed. All implants were well tolerated, but bone apposition was higher on calcium phosphate bone cements than on poly(methyl methacrylate) cement. Incorporation of collagen showed no evidence of inflammatory or immune reactions. Presence of positive tartrate-resistant acid phosphatase staining within cracks formed in calcium phosphate bone cements suggested active osteoclasts were present within the implants and were actively remodelling within the cements. Bone growth was also observed within these cracks. These findings confirm the biological advantages of calcium phosphate bone cements over poly(methyl methacrylate) and, coupled with previous work on enhancement of mechanical properties through collagen incorporation, suggest collagen-calcium phosphate bone cement composite may offer an alternative to calcium phosphate bone cements in applications where low setting times and higher mechanical stability are important.     

In vivo bone regeneration with injectable chitosan/hydroxyapatite/collagen composites and mesenchymal stem cells

For reconstruction of irregular bone defects, injectable biomaterials are more appropriate than the preformed biomaterials. We herein develop a biomimetic in situ-forming composite consisting of chitosan (CS) and mineralized collagen fibrils (nHAC), which has a complex hierarchical structure similar to natural bone. The CS/nHAC composites with or without mesenchymal stem cells (MSCs) are injected into cancellous bone defects at the distal end of rabbit femurs. Defects are assessed by radiographic, histological diagnosis and Raman microscopy until 12 weeks. The results show that MSCs improve the biocompatibility of CS/nHAC composites and enhance new bone formation in vivo at 12 weeks. It can be concluded that the injectable CS/nHAC composites combined with MSCs may be a novel method for reconstruction of irregular bone defects.     

Bone regeneration using an injectable calcium phosphate/autologous iliac crest bone composites for segmental ulnar defects in rabbits

Background Treatment of segmental bone loss remains a challenge in skeletal repair. A major therapeutic goal is the development of implantable materials that will promote bone regeneration. Objective We evaluate bone regeneration in grafts containing different concentrations autologous iliac crest bone (ACB) particles, carried in a new injectable calcium phosphate cement (CPC), in ulnar bone defects in rabbits. Methods Large upper-mid-diaphyseal defects (10 mm) were created in the left ulnae of 60 skeletally mature New Zealand white rabbits. ACB concentrations of 0, 25, 50, 75, and 100% (by volume) in CPC were used to fill operated sites. Defect bridging was monitored by serial radiography at 4, 8, and 12 weeks post-operation. Samples were then examined histologically and by manual palpation to determine the extent of new bone formation. Results At 4 weeks, we observed more elaborate structures and extensive absorption in ulnae treated with mixtures containing low concentrations of ACB (such as 0% and 25% volume of ACB/CPC), compared with those treated with mixtures containing high concentrations of ACB (such as 75% and 100% volume of ACB/CPC). At 8 weeks, histomorphometry revealed increased trabecular area and volume in the group treated with high ACB concentrations compared with those treated with low ACB concentrations. At 12 weeks, complete cortical bridging and regeneration of marrow space were detected in groups treated with high concentrations of ACB, and the amount of new bone regeneration was greater in these groups than in those treated with low ACB concentrations. Conclusions Treatment of rabbit ulnar defects with injectable CPC carrying an optimized concentration of ACB particles can lead to cortical bridging and bone marrow regeneration within 12 weeks.     

In vivo study of a bioactive nanoparticle-gelatin composite scaffold for bone defect repair in rabbits

The purpose is to study the in vivo bioactivity of this scaffold and verify its ability to simulate the characteristics of cancellous bone. Twenty-four adult New Zealand white rabbits were divided into three groups. Bone defects above the femoral condylar of both sides were created. A newly designed bioactive nanoparticle–gelatin composite scaffold was implanted to the experimental side, while the control side was left without implantation. The repair of bone defect was monitored by X-ray examination, gross observation, Micro-CT examination and histological observation of the area of bone defect 4, 8 and 12 weeks after surgery. There was void of new bone tissue in medullary cavity in the bone defect area of the control side. In the experimental side, the composite scaffold displayed excellent biodegradability, bioactivity and cyto-compatibility. With the time laps, new bone tissue grew from the edge to center as revealed by both Micro-CT image and staining biopsy, which complies with the “creeping substitution” process. The mechanical properties of the newly designed bioactive nanoparticle–gelatin composite scaffold and the 3-D structure of new bone tissue are comparable to the surrounding cancellous bones. This newly developed bioactive nanoparticle–gelatin composite scaffold possesses good biocompatibility and in vivo osteogenic capability for bone defect repair. It may be a promising artificial bone grafts.     

A novel photothermally controlled multifunctional scaffold for clinical treatment of osteosarcoma and tissue regeneration

After an osteosarcoma excision, recurrence, large bone defects, and soft tissue injury are significant challenges for clinicians. Conventional treatment by implanting bone replacement materials can induce bone regeneration after surgery, but this does not prevent bleeding, promote soft tissue repair, or help destroy the residual tumor cells. We attempted to develop a new multifunctional scaffold, with the clinical goals of facilitating tumor cell death through thermal ablation and promoting osteogenesis. Accordingly, we first investigated the effect of nano-hydroxyapatite/graphene oxide (nHA/GO) composite particles with different proportions on human osteosarcoma cells (HOS), pre-osteoblastic MC3T3-E1 cells, and human bone marrow mesenchymal stem cells (hBMSC) with or without 808-nm near-infrared (NIR) light irradiation. Next, we fabricated a novel temperature-controlled multifunctional nano-hydroxyapatite/graphene oxide/chitosan (nHA/GO/CS) scaffold, which can effectively kill human osteosarcoma cells under 808-nm NIR irradiation by reaching a temperature of 48 °C and further promote osteogenesis of hBMSC at 42 ± 0.5 °C in coordination with nHA. This scaffold demonstrates the best post-operative bone volume/tissue volume (BV/TV) ratio performance (20.36%) 8 weeks after scaffold implantation in the cranial defects of rats. Further exploration has revealed that NIR irradiation may promote the osteogenesis of hBMSC with the addition of nHA by enhancing the BMP2/Smad signaling pathway. Further, this scaffold has a good hemostatic effect and facilitates soft tissue repair under irradiation. This novel photothermally controlled multifunctional scaffold, which not only kills human osteosarcoma cells but also facilitates tissue regeneration, is a promising clinical tool for treating tissue injuries from an osteosarcoma resection.     

A simple method of cartilage regeneration using a new polymerizing system: ultrastructural characteristics of the repair tissue

The ultrastructural characteristics of the repair tissue in large articular cartilage defects, filled with a heterocyclic polymerizing system were studied using transmission electron microscopy (TEM) and energy dispersive micro-analysis (EDMA). By six weeks post-implantation, the defects were resurfaced with predominantly hyaline-like articular cartilage. Chondrocytes in both the superficial and deep zones of the repair tissue were highly productive, secreting large amounts of proteoglycans, into a well-organized, rich in collagen fibrils, extracellular matrix. By contrast, in the repair tissue of the defects treated without the biomaterial, proteoglycan synthesis was less and the structure of the matrix was inferior. We conclude that the polymer enhances both chondrocyte metabolism and matrix organization, thus improving the quality of the repair tissue in articular cartilage defects.     

Enhancement of nerve regeneration along a chitosan conduit combined with bone marrow mesenchymal stem cells

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration with different biomaterials. Nerve autografting is the most common surgical procedure currently used to repair nerve defects as a gold standard. To address the disadvantages of limited availability of donor nerves and donor site morbidity, we have fabricated chitosan conduits and seeded them combined with bone marrow mesenchymal stem cells (BMSCs) as an alternative. The conduits were tested for efficacy in bridging the critical gap (8 mm) in sciatic nerves of adult rats, which including sciatic nerve function index (SFI), ethology observation, histologic detection, immunohistochemistry detection. The BMSCs were tested for survival rate and differentiation by fluorescence labeling. Six weeks after operation, the SFI, average regenerated fiber density, and fiber diameter in nerves bridged with BMSCs were similar to those treated with autograft, but significantly higher than those bridged with chitosan conduits only (P < 0.05) because of the differentiation of BMSCs. Evidence is thus provided to support the effect of using multi-channel chitosan conduits seeded with BMSCs to treat critical defects in peripheral nerves. This provides the basis to pursue chitosan and BMSCs combination is an effective method to improve the nerve healing, which may be used as an alternative to the conventional nerve autografts.     

Repair of rabbit femoral condyle bone defects with injectable nanohydroxyapatite/chitosan composites

Repair of massive bone loss remains a challenge to the orthopaedic surgeons. Autologous and allogenic bone grafts are choice for bone reconstructive surgery, but limited availability, risks of transmittable diseases and inconsistent clinical performances have prompted the development of tissue engineering. In the present work, the bone regeneration potential of nanohydroxyapatite/chitosan composite scaffolds were compared with pure chitosan scaffolds when implanted into segmental bone defects in rabbits. Critical size bone defects (6 mm diameter, 10 mm length) were created in the left femoral condyles of 43 adult New Zealand white rabbits. The femoral condyle bone defects were repaired by nanohydroxyapatite/chitosan compositions, pure chitosan or left empty separately. Defect-bridging was detected by plain radiograph and quantitative computer tomography at eight and 12 weeks after surgery. Tissue samples were collected for gross view and histological examination to determine the extent of new bone formation. Eight weeks after surgery, more irregular osteon formation was observed in the group treated with nanohydroxyapatite/chitosan composites compared with those treated with pure chitosan. 12 weeks after surgery, complete healing of the segmental bone defect was observed in the nanohydroxyapatite/chitosan-group, while the defect was still visible in the chitosan-group, although the depth of the defect had diminished. These observations suggest that the injectable nanohydroxyapatite/chitosan scaffolds are potential candidate materials for regeneration of bone loss.     

Carbon nanohorns accelerate bone regeneration in rat calvarial bone defect

A recent study showed that carbon nanohorns (CNHs) have biocompatibility and possible medical uses such as in drug delivery systems. It was reported that some kinds of carbon nanomaterials such as carbon nanotubes were useful for bone formation. However, the effect of CNHs on bone tissue has not been clarified. The purpose of this study was to evaluate the effect of CNHs on bone regeneration and their possible application for guided bone regeneration (GBR). CNHs dispersed in ethanol were fixed on a porous polytetrafluoroethylene membrane by vacuum filtration. Cranial defects were created in rats and covered by a membrane with/without CNHs. At two weeks, bone formation under the membrane with CNHs had progressed more than under that without CNHs and numerous macrophages were observed attached to CNHs. At eight weeks, there was no significant difference in the amount of newly formed bone between the groups and the appearance of macrophages was decreased compared with that at two weeks. Newly formed bone attached to some CNHs directly. These results suggest that macrophages induced by CNHs are related to bone regeneration. In conclusion, the present study indicates that CNHs are compatible with bone tissue and effective as a material for GBR.     

Effects of porous beta-tricalcium phosphate-based ceramics used as an E. coli-derived rhBMP-2 carrier for bone regeneration

Recombinant human bone morphogenetic protein-2 (rhBMP-2) requires carriers for clinical effectiveness. In this study, whether porous beta-tricalcium phosphate (β-TCP)-based ceramics are ideal carriers for rhBMP-2 was investigated. Hydroxyapatite (HA), β-TCP, TCP/HA (80 %/20 %), HA with rhBMP-2, TCP with rhBMP-2, and TCP/HA (80 %/20 %) with rhBMP-2 were manufactured by a sponge method with a pore size of 300 μm or more and macro-porosity of 83 %. The alkaline phosphatase (ALP) activity and ALP expression of the cells with 100 % β-TCP granules were more increased than the those of cells with 100 % HA and TCP/HA (80 %/20 %) at the baseline or when treated with 15 ng/ml of rhBMP-2. In an SD rat calvarial defect model, new bone formation was evidently shown in the TCP 100 %-rhBMP-2 and TCP/HA (80 %/20 %)-rhBMP-2 groups, showing that the most affected area was filled with newly-formed bone, that the percent bone volume and trabecular number were larger when compared to the groups without rhBMP-2 treatment at both 4 and 8 weeks after surgery using micro-CT and histology. Porous TCP-based ceramic granules enhanced the osteoblastic differentiation in the hMSC system when treated with 15 ng/ml of rhBMP-2 and accelerated bone-healing by trabecular number in a rat calvarial defect model. Thus, in this study it was proposed that TCP-based ceramics might be useful carriers of rhBMP-2.     

LvBMP-2 gene-modified BMSCs combined with calcium phosphate cement scaffolds for the repair of calvarial defects in rats

The study aims to evaluate the effect of bone marrow stromal cells (BMSCs) expressing bone morphogenic protein-2 (BMP-2) mediated by lentiviral (Lv) gene transduction combined with calcium phosphate cement (CPC) scaffolds for the repair of critical size calvarial defects in rats. BMSCs derived from Fisher 344 rats were transduced with LvBMP-2 or lentivirus encoding enhanced green fluorescent protein (LvEGFP) in vitro. Obvious osteogenic differentiation of BMSCs in the LvBMP-2 group was demonstrated by alkaline phosphatase staining and alizarin red staining. Enzyme-linked immunosorbent assay results show that LvBMP-2 gene expression in vitro can last for at least 8 weeks. Gene-transduced or untransduced BMSCs were seeded onto CPC scaffolds to repair rat calvarial defects with a diameter of 5 mm. Scanning electron microscope analysis indicated that porous CPC scaffolds facilitated initial adhesion and spreading of BMSCs onto its surface. Calvarial defects were successfully repaired with LvBMP-2-transduced BMSCs/CPC constructs 8 weeks postoperatively. The percentage of new bone formation in the LvBMP-2 group was significantly higher than in other control groups. Lentiviral mediated BMP-2 gene therapy together with CPC scaffolds can be used successfully in calvarial repair and bone regeneration.     

A hydrophylic polymer system enhanced articular cartilage regeneration in vivo

This study describes a new method for the repair of large articular cartilage defects in the knee joint and compares the effect of two polymer systems on the quality of the repair tissue. The two systems are a newly developed hydrophylic system, based on poly-ethyl-methacrylate (PEMA) polymer and tetra-hydro-furfuryl-methacrylate (THFMA) monomer and the conventional bone cement polymer system, based on poly-methyl-methacrylate (PMMA) polymer and methyl-methacrylate (MMA) monomer. Thirty adult Sandy-lop rabbits were used. Both knees were operated on in each animal, the one defect received either PEMA/THFMA or conventional bone cement and the contralateral defect received no biomaterial (control group). Femora were retrieved at six weeks and the repair tissue was studied by histology, histochemistry and immuno-histochemistry. PEMA/THFMA enhanced the quality of the repair significantly (p<0.0001). By six weeks hyaline-like articular cartilage was the predominant tissue covering the defects and it was fully integrated with the surrounding normal articular cartilage. Immuno-localization showed cartilage components, including collagen type II, distributed evenly throughout its matrix. PMMA/MMA on the other hand did not improve significantly the repair tissue, which was predominately fibro-cartilaginous, poorly bonded to the adjacent normal articular cartilage. The method of implantation is simple and easily reproducible and the new polymer has been well-accepted by the rabbits.     

Si-doping bone composite based on protein template-mediated assembly for enhancing bone regeneration

Bio-inspired hybrid materials that contain organic and inorganic networks interpenetration at the molecular level have been a particular focus of interest on designing novel nanoscale composites. Here we firstly synthesized a series of hybrid bone composites, silicon-hydroxyapatites/silk fibroin/collagen, based on a specific molecular assembled strategy. Results of material characterization confirmed that silicate had been successfully doped into nano-hydroxyapatite lattice. In vitro evaluation at the cellular level clearly showed that these Si-doped composites were capable of promoting the adhesion and proliferation of rat mesenchymal stem cells (rMSCs), extremely enhancing osteoblastic differentiation of rMSCs compared with silicon-free composite. More interestingly, we found there was a critical point of silicon content in the composition on regulating multiple cell behaviors. In vivo animal evaluation further demonstrated that Si-doped composites enabled to significantly improve the repair of cranial bone defect. Consequently, our current work not only suggests fabricating a potential bone repair materials by integrating element-doping and molecular assembled strategy in one system, but also paves a new way for constructing multi-functional composite materials in the future.     

Material design of bioabsorbable inorganic/organic composites for bone regeneration

Two new bioabsorbable inorganic/organic composite materials were developed for bone regeneration. One material used was beta-TCP/PLGC in which poly(L-lactide-co-glycolide-co-epsilon-caprolactone) and beta-tricalcium phosphate were used as the matrix and filler, respectively. The other material used was HAp/Col-a soft nanocomposite of hydroxyapatite and type I collagen. Using these composites, two bone implants were designed. The efficacy of these implants was investigated by applying them to the critical-sized bone defects that were created in the canine tibia. Although no tissue engineering techniques such as application of growth factors or stem cells was utilized, successful healing was observed. These results suggested that bone regeneration in the critical-sized defects is possible without the use of growth factors or stem cells if the materials and the bone implants are suitably designed.     

Interfacial and biological properties of the gradient coating on polyamide substrate for bone substitute

Fabrication of bioactive and mechanical matched bone substitutes is crucial for clinical application in bone defects repair. In this study, nano-hydroxyapatite/polyamide (nHA/PA) composite was coated on injection-moulded PA by a chemical corrosion and phase-inversion technique. The shear strength, gradient composition and pore structure of the bioactive coating were characterized. Osteoblast-like MG63 cells were cultured on pure PA and composite-coated PA samples. The cells'' adhesion, spread and proliferation were determined using MTT assay and microscopy. The results confirm that the samples with the nHA/PA composite coating have better cytocompatibility and have no negative effects on cells. To investigate the in vivo biocompatibility, both pure PA and composite-coated PA cylinders were implanted in the trochlea of rabbit femurs and studied histologically, and the bonding ability with bone were determined using push-out tests. The results show that composite-coated implants exhibit better biocompatibility and the shear strength of the composite-coated implants with host bone at 12 weeks can reach 3.49 ± 0.42 MPa, which is significantly higher than that of pure PA implants. These results indicate that composite-coated PA implants have excellent biocompatibility and bonding abilities with host bone and they have the potential to be applied in repair of bone defects.     

磷酸钙骨水泥/明胶复合组织工程支架材料的制备及其结构与性能

磷酸钙骨水泥组织工程支架材料具有良好的生物相容性和骨传导性,是一种良好的骨组织工程支架材料,但是这种材料存在力学性能差的缺点,限制了它的应用.本文采用生物相容性良好的可降解明胶材料与磷酸钙骨水泥支架进行复合,制备出的明胶/磷酸钙骨水泥复合支架材料,其压缩强度可达3.7MPa,比复合前磷酸钙支架材料的强度提高了37倍,而且材料具有良好的柔韧性,适合用作为非承重部位骨组织缺损修复用组织工程支架材料.