The role of pore size on vascularization and tissue remodeling in PEG hydrogels

Vascularization is influenced by the physical architecture of a biomaterial. The relationship between pore size and vascularization has been examined for hydrophobic polymer foams, but there has been little research on tissue response in porous hydrogels. The goal of this study was to examine the role of pore size on vessel invasion in porous poly(ethylene glycol) (PEG) hydrogels. Vascularized tissue ingrowth was examined using three-dimensional cell culture and rodent models. In culture, all porous gels supported vascular invasion with the rate increasing with pore size. Following subfascial implantation, porous gels rapidly absorbed wound fluid, which promoted tissue ingrowth even in the absence of exogenous growth factors. Pore size influenced neovascularization, within the scaffolds and also the overall tissue response. Cell and vessel invasion into gels with pores 25-50 μm in size was limited to the external surface, while gels with pores larger pores (50-100 and 100-150 μm) permitted mature vascularized tissue formation throughout the entire material volume. A thin layer of inflammatory tissue was present at all PEG-tissue interfaces, effectively reducing the area available for tissue growth. These results show that porous PEG hydrogels can support extensive vascularized tissue formation, but the nature of the response depends on the pore size.     

血管化人工骨的体外构建和体内异位成骨

背景：血管化在骨形成和改建中起重要作用,目前大的组织块难以获得充足的氧气和营养供应,因此组织工程中就出现了需要适当血管化的问题。 目的：建立体外血管化人工骨模型并且体内异位成骨的实验体系。 方法：分离培养鼠骨髓间充质干细胞和肾血管内皮细胞,体外构建鼠骨髓间充质干细胞和鼠肾血管内皮细胞直接接触培养血管化人工骨组,以间接接触三维培养体系和两种细胞单独培养体系作为对照。体外通过测定蛋白质含量,碱性磷酸酶活性和骨钙素,分析骨髓间充质干细胞在不同混合培养模型中的成骨能力。建立鼠骨髓间充质干细胞和肾血管内皮细胞的三维培养体系,再将两种细胞直接接触组材料和骨髓间充质干细胞单独培养组的材料分别植入鼠左右腿肌肉内,通过软X射线摄影和苏木精-伊红染色检测分析不同植入方法的血管形成和成骨能力。 结果与结论：当两种细胞混合培养时,具有很好的细胞兼容性。两种细胞单独培养碱性磷酸酶活性和骨钙素较间接接触混合培养降低,而两种细胞混合培养时,体系中碱性磷酸酶活性和骨钙素水平增加。动物实验结果显示在混合培养体系中的骨髓间充质干细胞的成骨能力高于单独培养(P < 0.05)。体内实验表明在血管化人工骨中的软X射线骨密度,血管数量和新形成的骨量均高于对照组(P < 0.05)。提示在两种细胞混合培养时骨髓间充质干细胞的成骨能力可以被细胞因子和细胞膜蛋白质调节,和传统的人工骨比较,血管化人工骨有加速骨髓间充质干细胞分化,增加了局部的血管微循环生存率,以及加速成骨和更好的抗感染能力。     

Simultaneous in vivo comparison of bone substitutes in a guided bone regeneration model

A direct, simultaneous comparison of bone substitutes is hampered by the limited number of samples that can be tested simultaneously. The goal of this study was to establish a preclinical model for guided bone regeneration that offers testing of different bone substitutes in a one-wall defect situation. We show here that up to eight titanium hemispheres can be placed on the calvaria of minipigs. To establish our model, titanium hemispheres were filled with and without Bio-Oss, a deproteinized bovine bone mineral, Ostim, an aqueous paste of synthetic nanoparticular hydroxyapatite, and Osteoinductal, an oily calcium hydroxide suspension, before being positioned on the calvaria. After 6 and 12 weeks, titanium hemispheres were subjected to histological and histomorphometric analysis. We show here that bone filled approximately one-tenth of the area below the hemispheres which were left empty, indicating a critical size model for guided bone regeneration. In accordance with the documented osteoconductive properties of Bio-Oss and Ostim, titanium hemispheres were almost completely filled with bone. Moreover, the expected degradation profile of Bio-Oss and Ostim could be confirmed by histologic and histomorphometric analysis. Under the same conditions, Osteoinductal failed to exert osteoconductive properties, rather a progressive resorption of the host bone was observed. These results demonstrate that the preclinical model presented here is suitable to simultaneously compare bone substitutes with different material properties. Our model based on the titanium hemispheres allows evaluation of graft consolidation under standardized conditions thereby avoiding intra-individual variations.     

In vivo molecular evidence of delayed titanium implant osseointegration in compromised bone

Optimization of implant osseointegration in patients with reduced bone healing potential is a challenge remaining in implant dentistry. Identification of the genes that are modulated during implant osseointegration in normal versus osteopenic bone is needed to successfully address these pertinent clinical needs. The present study aimed to assess the initial and early molecular events following titanium implant installation in normal and compromised bone in a rat tibia model. Peri-implant tissue from a well-defined tissue regeneration compartment was analyzed at 2 and 7 days post-surgery for the expression of select markers of inflammation, angiogenesis, bone resorption and bone formation. Impaired bone was induced by hindlimb unloading and validated using μCT. The essential step of angiogenesis preceding bone regeneration was evidenced for the peri-implant setting in healthy bone. Compromised bone significantly affected the angiogenesis-osteogenesis coupling in the initial phase (2?days post-surgery), with altered expressions of Vegfa and Epas1 coinciding with downregulated expressions of Col1a1, Bmp2, Bmp4, Alpl and Bglap. At 7 days post-implantation, differences between normal and compromised peri-implant bone were no longer observed. This in vivo molecular evidence of delayed implant osseointegration in compromised bone reassert modern strategies in implant development, such as surface modifications and bioengineered approaches, to improve implant osseointegration in compromised conditions.     

Of the in vivo behavior of calcium phosphate cements and glasses as bone substitutes

The use of injectable self-setting calcium phosphate cements or soluble glass granules represent two different strategies for bone regeneration, each with distinct advantages and potential applications. This study compares the in vivo behavior of two calcium phosphate cements and two phosphate glasses with different composition, microstructure and solubility, using autologous bone as a control, in a rabbit model. The implanted materials were alpha-tricalcium phosphate cement (cement H), calcium sodium potassium phosphate cement (cement R), and two phosphate glasses in the P(2)O(5)-CaO-Na(2)O and P(2)O(5)-CaO-Na(2)O-TiO(2) systems. The four materials were osteoconductive, biocompatible and biodegradable. Radiological and histological studies demonstrated correct osteointegration and substitution of the implants by new bone. The reactivity of the different materials, which depends on their solubility, porosity and specific surface area, affected the resorption rate and bone formation mainly during the early stages of implantation, although this effect was weak. Thus, at 4 weeks the degradation was slightly higher in cements than in glasses, especially for cement R. However, after 12 weeks of implantation all materials showed a similar degradation degree and promoted bone neoformation equivalent to that of the control group.     

In vivo analysis of biocompatibility and vascularization of the synthetic bone grafting substitute NanoBone

One of the major challenges in the application of bone substitutes is adequate vascularization and biocompatibility of the implant. Thus, the temporal course of neovascularization and the microvascular inflammatory response of implants of NanoBone (fully synthetic nanocrystalline bone grafting material) were studied in vivo by using the mouse dorsal skinfold chamber model. Angiogenesis, microhemodynamics, and leukocyte-endothelial cell interaction were analyzed repetitively after implantation in the center and in the border zone of the implant up to 15 days. Both NanoBone granules and plates exhibited high biocompatibility comparable to that of cancellous bone, as indicated by a lack of venular leukocyte activation after implantation. In both synthetic NanoBone groups, signs of angiogenesis could be observed even at day 5 after implantation, whereas granules showed higher functional vessel density compared with NanoBone plates. The angiogenic response of the cancellous bone was markedly accelerated in the center of the implant tissue. Histologically, implant tissue showed an ingrowth of vascularized fibrous tissue into the material combined with an increased number of foreign-body giant cells. In conclusion, NanoBone, particularly in granular form, showed high biocompatibility and high angiogenic response, thus improving the healing of bone defects. Our results underline that, beside the composition and nanostructure, the macrostructure is also of importance for the incorporation of the biomaterial by the host tissue.     

In vivo evaluation of resorbable bone graft substitutes in mandibular sockets of the beagle

Hydroxyapatite (Ca(10) (PO(4) )(6) (OH)(2) ), with its high biocompatibility and good bioaffinity, stimulates osteoconduction and is slowly replaced by the host bone after implantation. However, clinical use of HA as a bone substitute has proved problematic. It is difficult to prevent dispersion of the HA granules and to mold the granules into the desired shape. Calcium sulfate as a bone graft substitute is rapidly resorbed in vivo releasing calcium ions, but fails to provide a long-term, three-dimensional framework to support osteoconduction. The setting properties of calcium sulfate, however, allow it to be applied in a slurry form, making it easier to handle and apply in different situations. This study examines the in vivo response of a (Hydroxyapatite, apatitic phase)/calcium sulfate dehydrate (CSD) composite using different ratios in the mandibular premolar sockets of the beagle. The HA (AP)/CSD composite materials prepared in ratios of 30/70, 50/50, and 70/30 were implanted into the mandibular premolar sockets for 5 and 10 weeks. The control socket was empty. The authors compared the radiographic properties and the changes in height and width of the mandibular premolar sockets in the beagle. The composite graft in the 30/70 ratio had the best ability to form new bones. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2726-2731, 2012.     

The promotion of the vascularization of decalcified bone matrix in vivo by rabbit bone marrow mononuclear cell-derived endothelial cells

The neovascularization of bone grafting represents an important challenge in bone regeneration. Prevascularization of tissue-engineered bone using endothelial cells (ECs) in vitro sheds light on accelerating the vascularization of bone replacements. In the present study, decalcified bone matrix (DBM) was prevascularized by seeding fibrin gels with ECs that are derived from rabbit bone marrow mononuclear cells (BMMNCs). The compound was then transplanted autologously into bone defects of rabbits to observe the vascularization in vivo. At 2, 4 and 8 weeks after grafting, the microvessel density of new bone tissues was significantly higher in the experimental group than in the control group (P < 0.05), which suggests that prevascularization of BMMNC-derived cells may be suitable for improving vascularization in tissue-engineered bone.     

Injectable nanocrystalline hydroxyapatite paste for bone substitution: in vivo analysis of biocompatibility and vascularization

The nanocrystalline hydroxyapatite paste Ostim represents a fully degradable synthetic bone substitute for the filling of bone defects. Herein, we investigated in vivo the inflammatory and angiogenic host tissue response to this biomaterial after implantation. For this purpose, Ostim was implanted into the dorsal skinfold chambers of Syrian golden hamsters. The hydroxyapatite ceramic Cerabone and isogeneic transplanted cancellous bone served as controls. Angiogenesis, microhemodynamics, microvascular permeability, and leukocyte-endothelial cell interaction of the host tissue were analyzed over 2 weeks using intravital fluorescence microscopy. Ostim exhibited good biocompatibility comparable to that of Cerabone and cancellous bone, as indicated by a lack of venular leukocyte activation after implantation. Cancellous bone induced a more pronounced angiogenic response and an increased microvessel density when compared with the synthetic bone substitutes. In contrast to Cerabone, however, Ostim showed a guided neovascularization directed toward areas of degradation. Histology confirmed the ingrowth of proliferating vascularized tissue into the hydroxyapatite paste at sites of degradation, while the hydroxyapatite ceramic was not pierced by new microvessels. Thus, Ostim represents an injectable synthetic bone substitute, which may optimize the conditions for the formation of new bone at sites of bone defects by supporting a guided vascularization during biodegradation.     

Effect of hydroxyapatite coating crystallinity on dissolution and osseointegration in vivo

Hydroxyapatite (HA) coating with different crystallinities were deposited by plasma spraying and vapor-flame treatment process. Their crystallinities are about 55 and 98%, respectively. These coatings were implanted in cortical bone, muscle, and marrow of dogs. The dissolution and osseointegration behavior were evaluated by scanning electron microscope (SEM) observation histological analysis. The results obtained indicated that after implanted in muscle, a bone-like apatite layer was formed on the surface of as-sprayed coating, which was not observed on the surface of the treated coating. The as-sprayed coating has the ability to induce new bone formation on its surface after implanted in marrow. In contrast, the treated coating displays a limited bone bioactivity. The vapor-flame process diminishes the short-term osseointegration properties of the HA coating, but no significant affection was found after three months implantation. Either in muscle or in cortical bone, treated coating exhibits higher stability than the as-sprayed coating, in some conditions.     

In vivo evaluation of resorbable bone graft substitutes in a rabbit tibial defect model

Calcium sulfate as a bone graft substitute is rapidly resorbed in vivo releasing calcium ions but fails to provide long-term three-dimensional framework to support osteoconduction. The setting properties of calcium sulfate however allow it to be applied in a slurry form making it easier to handle and apply in different situations. This study examines the in vivo response of calcium sulfate alone and as a carrier for a coralline hydroxyapatite in an established bilateral corticocancellous defect model in rabbits. Defects were filled flush to the anterior cortex with a resorbable porous ceramic alone and in combination with calcium sulfate slurry, calcium sulfate slurry alone or calcium sulfate pellets and examined at time points up to 52 weeks. Specimens where assessed using Faxitron X-ray, light and electron microscopy. Calcium sulfate in either slurry or pellet form does indeed support new bone formation alone however, complete filling of the bone defect is not observed. Calcium sulfate in slurry form does however improve the surgical handling of particulate bone graft substitutes such as Pro Osteon 200 R, which remained as an osteoconductive scaffold for up to 52 weeks and may have played an important role in the ultimate closure of the cortical windows.     

Effect of titanium carbide coating on the osseointegration response in vitro and in vivo

Titanium has limitations in its clinical performance in dental and orthopaedic applications. This study describes a coating process using pulsed laser deposition (PLD) technology to produce surfaces of titanium carbide (TiC) on titanium substrates and evaluates the biological response both in vitro and in vivo. X-ray photoelectron spectroscopy (XPS) analysis revealed the presence of 18.6-21.5% TiC in the surface layer, accompanied by oxides of titanium 78.5-81.4% in the following concentrations: 11.1-13.0% Ti(2)O(3), 50.8-55.8% TiO(2), 14.5-14.7% TiO. Expression of genes central to osteoblast differentiation (alkaline phosphatase, A2 pro-collagen type 1, osteocalcin, BMP-4, TGFbeta and Cbfa-1) were up-regulated in all cell lines (primary human osteoblasts, hFOB1.19 and ROS.MER#14) grown on TiC compared with uncoated titanium when measured by semiquantitative PCR and real time-PCR, whilst genes involved in modulation of osteoclastogenesis and osteoclast activity (IL-6 and M-CSF) were unchanged. Bone density was shown to be greater around TiC-coated implants after 2 and 4 weeks in sheep and both 4 and 8 weeks in rabbits compared to uncoated titanium. Rapid bone deposition was demonstrated after only 2 weeks in the rabbit model when visualized with intravital staining. It is concluded that coating with TiC will, in comparison to uncoated titanium, improve implant hardness, biocompatibility through surface stability and osseointegration through improved bone growth.     

Stimulation of peri-implant vascularization with bone marrow-derived progenitor cells: monitoring by in vivo EPR oximetry

The poorly vascularized fibrous capsule that develops around implantable biomedical devices (for drug delivery, biosensors, etc.) severely limits their applications. We tested the hypotheses that co-implantation of bone marrow-derived progenitor cells could stimulate the vascularization of implants. To assess the presence of functional peri-implant microvasculature, we developed a novel model of implanted device containing an oxygen (O(2))-sensing spin probe (detectable using electron paramagnetic resonance) placed inside a nanoporous filter-limited capsule. These devices were implanted subcutaneously in C57/Bl6 mice alone, with the addition of a Matrigel plug in front of the filter, or with the addition of Matrigel containing equal proportions of c-kit(+) and stem cell antigen-1(+) bone marrow-derived cells. Implants partial pressure of O(2) (pO(2)) were recorded non-invasively and periodically for up to 10 weeks. Tissue surrounding the implants was collected for immunohistochemistry. Initially, there were no differences in pO(2) between the experimental groups. After 3 weeks, the devices supplied with progenitor cells showed more than twice the O(2) concentrations as controls. This difference remained significant for 4 more weeks and then started to decrease slightly, still being 6 mmHg higher than in the controls at 10 weeks post-implantation. Collagen deposition was detected around the control implants, along with F4/80-positive macrophages and giant cells. In the plugs collected from the cell treatment group, we found an active process of adipogenesis, accompanied by neovascularization, and a highly vascularized adipose layer surrounding the implants. In conclusion, we successfully developed a cell therapy-type strategy to maintain vascularization around implanted devices using co-administration of bone marrow-derived progenitor cells, and we demonstrated a novel O(2)-sensing method to functionally monitor neovascularization in vivo.     

血管化在骨组织工程中的应用

随着骨缺损修复治疗方法的不断完善发展,组织工程化人工骨被逐渐应用于此治疗,但随着组织工程技术的提高,血管化应用于人工骨将代替单纯应用人工骨,并被认为是将来较理想的修复方法。本文将对骨组织血管化的机理,血管化在骨组织工程中的作用和具体应用进行综述。     

Hydroxyapatite-polyethylene composites for bone substitution: effects of ceramic particle size and morphology

Synthetic hydroxyapatite particles of two median sizes and different morphologies have been used to manufacture hydroxyapatite reinforced high density polyethylene composites (HAPEX^TM) for medical applications. The effects of hydroxyapatite particle size on properties of the resultant composites were investigated using various techniques. It was found that composites with smaller hydroxyapatite particles had higher torsional modulus, tensile modulus and tensile strength, but lower strain to failure. Examination of fracture surfaces revealed that only a mechanical bond existed between the filler and the matrix. It was shown that dynamic mechanical analysis is useful in studying the viscoelastic behaviour of the composite.     

Comparative effects of scaffold pore size, pore volume, and total void volume on cranial bone healing patterns using microsphere-based scaffolds

Bony craniofacial deficits resulting from injury, disease, or birth defects remain a considerable clinical challenge. In this study, microsphere-based scaffold fabrication methods were use to study the respective effects of scaffold pore size, open pore volume, and total void volume fraction on osseous tissue infiltration and bone regeneration in a critical size rat cranial defect. To compare the healing effects of these parameters, three different scaffolds types were fabricated: solid 100 microm spheres, solid 500 microm spheres, and hollow 500 microm spheres. These constructs were implanted into surgically created rat calvarial defects. By 90-days post op, results of micro computed tomography (CT) analysis showed that all scaffolds generated similar amounts of new bone which was significantly greater than untreated controls. Interestingly, the spatial distribution of new bone within the defect area varied by scaffold group. MicroCT and histological analysis demonstrated healing restricted to the dural side in the hollow 500 microm group, whereas the solid 500 microm group demonstrated healing along the dural side and within the center of the defect. Solid 100 microm groups demonstrated healing along the dural layer, periosteal layer, and within the center of the defect. These results suggest that pore size and closed void volume may both play important roles in scaffold degradation patterns and associated bone healing.     

Development of gelatin-chitosan-hydroxyapatite based bioactive bone scaffold with controlled pore size and mechanical strength

Hydroxyapatite–chitosan/gelatin (HA:Chi:Gel) nanocomposite scaffold has potential to serve as a template matrix to regenerate extra cellular matrix of human bone. Scaffolds with varying composition of hydroxyapatite, chitosan, and gelatin were prepared using lyophilization technique where glutaraldehyde (GTA) acted as a cross-linking agent for biopolymers. First, phase pure hydroxyapatite–chitosan nanocrystals were in situ synthesized by coprecipitation method using a solution of 2% acetic acid dissolved chitosan and aqueous solution of calcium nitrate tetrahydrate [Ca(NO₃)₂,4H₂O] and diammonium hydrogen phosphate [(NH₄)₂H PO₄]. Keeping solid loading constant at 30 wt% and changing the composition of the original slurry of gelatin, HA–chitosan allowed control of the pore size, its distribution, and mechanical properties of the scaffolds. Microstructural investigation by scanning electron microscopy revealed the formation of a well interconnected porous scaffold with a pore size in the range of 35–150 μm. The HA granules were uniformly dispersed in the gelatin–chitosan network. An optimal composition in terms of pore size and mechanical properties was obtained from the scaffold with an HA:Chi:Gel ratio of 21:49:30. The composite scaffold having 70% porosity with pore size distribution of 35–150 μm exhibited a compressive strength of 3.3–3.5 MPa, which is within the range of that exhibited by cancellous bone. The bioactivity of the scaffold was evaluated after conducting mesenchymal stem cell (MSC) – materials interaction and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay using MSCs. The scaffold found to be conducive to MSC’s adhesion as evident from lamellipodia, filopodia extensions from cell cytoskeleton, proliferation, and differentiation up to 14 days of cell culture.     

Bioactivity and osseointegration study of calcium phosphate ceramic of different chemical composition

Synthetic hydroxyapatite (HA) and tricalcium phosphate (TCP) are promising bone-substitute materials in the orthopaedic and dental fields, as their chemical composition is similar to that of bone. This study investigated the osseointegration performance of carbonated biphasic calcium phosphate (CBCP) ceramics containing carbonated hydroxyapatite and tricalcium phosphate prepared by microwave irradiation, in femoral defects of dogs. The defects were created as 3-mm holes on the lateral aspect of femur and filled with the implant material. The serum was collected postoperatively and biochemical assays for alkaline phosphatase activity levels were carried out. The animals' defective sites were radiographed at 4, 8, and 12 weeks. The radiographic results showed that the process of ossification started after 4 weeks and the defect was completely filled with new bone after 8 weeks. Histological examination of the tissue showed the osteoblastic activity inducing the osteogenesis in the defect. The complete haversian system with osteoblastic and osteoclastic activity and bone remodelling process were observed after 12 weeks. The alkaline phosphatase activity levels also correlated with the formation of osteoblast cells. This calcium phosphate ceramic has proved to work well as a biocompatible implant and as an osteoconductive and osteoinductive material for the filling of bone defects.     

Mediation of bone ingrowth in porous hydroxyapatite bone graft substitutes

Previous investigations have shown that both the early biological response and the mechanical properties of a porous hydroxyapatite bone graft substitute are highly sensitive to its pore structure. The objective of this study was to evaluate whether the pore structure continued to influence bone integration in the medium to long term. Two screened batches of porous hydroxyapatite (PHA) designated as batch A and batch B, with porosities of approximately 60 and 80%, respectively, were selected for this study and implanted for periods of 5, 13, and 26 weeks into the lower femur of New Zealand White rabbits. Histomorphometric analysis of the absolute volume of bone ingrowth within batch A and B implants from 5 to 26 weeks showed that the absolute volume of bone ingrowth was consistently lower in batch A (10-21%), compared to batch B implants (24-31%). However, when the volume of bone ingrowth was normalised for the available pore space, this difference was reduced (23-47% and 32-42% for batches A and B, respectively). These observations suggest that differences in the volume of bone ingrowth initially depended on pore interconnectivity rather than pore size, whereas the volume or morphology of the PHA influenced the volume and morphology of bone ingrowth at later time points. Compression testing showed that bone ingrowth had a strong reinforcing effect on PHA bone graft substitutes, and a strong correlation was identified between mechanical properties and the absolute volume of ingrowth for both batches A and B. Furthermore, at 13 and 26 weeks, there was no significant variation in the ultimate compressive strength of integrated batch A and B implants. This similarity in ultimate mechanical properties indicated that the absolute volume of ingrowth may be mediated by the PHA structure through its impact on the dynamics of the local biomechanical environment. The results of push-out testing showed that fixation of PHA bone graft substitutes was independent of density within the range studied, with no significant difference in the interfacial shear stress between batches A and B at each time point throughout the study.     

骨血管形成机制及功能的研究进展

骨血管系统不仅为高代谢的骨细胞提供氧气与营养物质,也参与骨形成与骨再生过程。骨组织毛细血管可分为两种亚型,它们具有不同的形态和生理特点。内皮细胞与骨细胞通过多种信号通路沟通,血管形成与骨形成也具有协同作用。对骨血管结构及血管形成信号通路的探索有助于骨折愈合临床治疗的发展。