Use of natural coralline biomaterials as reinforcing and gas-forming agent for developing novel hybrid biomatrices: microarchitectural and mechanical studies

This paper describes the first attempt in fabrication of three-dimensional macroporous composites of chitosan and natural coralline material with pore sizes of 300-400 microm, exceeding the upper pore size limit of 250 microm obtained with freeze-dried chitosan-based scaffolds. Natural coral particulates of less than 20 microm, which is mainly composed of calcium carbonate (CaCO3), was simultaneously used as reinforcing phase and gas-forming agent to obtain a structure with large pores and improved mechanical and biological properties. The reaction between the coralline material and the acidic chitosan polymer solvent, which produced carbon dioxide, was rapidly stopped by the subsequent thermally induced phase separation technique, leaving coralline particulates in the polymeric structure. Scaffolds containing five different proportions of coralline material (0, 25, 50, 75, and 100 wt%) were investigated. The coralline-chitosan weight ratio was studied for its effects on the physical properties of the scaffolds. The relation between scaffold microarchitecture and mechanical properties was assessed with scanning electron microscope (SEM), along with micro-CT imaging and compression testing. The scaffolds were used in bone marrow cell culturing experiments to assess the effect of composition on cell behavior through cell-material interaction and morphological observation by SEM. Higher coralline concentration increased the pore wall thickness and favored large pore formation. Varying the coralline particulate to chitosan polymer ratio from 0 to 75 wt% increased the average pore size from 80 microm to 400 microm while the porosity decreased from 91% to 78%. The compressive modulus was improved proportionally with the coralline content, and the 75 wt% composites had a significantly higher modulus than other chitosan-based scaffold groups. More cells were observed on scaffolds with higher coralline content. The cell culture experiments indicated that the scaffolds containing coralline material might have a high cell affinity, since it allowed fast cell attachment and spreading.     

Porous PEOT/PBT scaffolds for bone tissue engineering: preparation,characterization, and in vitro bone marrow cell culturing

The preparation, characterization, and in vitro bone marrow cell culturing on porous PEOT/PBT copolymer scaffolds are described. These scaffolds are meant for use in bone tissue engineering. Previous research has shown that PEOT/PBT copolymers showed in vivo degradation, calcification, and bone bonding. Despite this, several of these copolymers do not support bone marrow cell growth in vitro. Surface modification, such as gas-plasma treatment, is needed to improve the in vitro cell attachment. Porous structures were prepared using a freeze-drying and a salt-leaching technique, the latter one resulting in highly porous interconnected structures of large pore size. Gas-plasma treatment with CO(2) generated a surface throughout the entire structure that enabled bone marrow cells to attach. The amount of DNA was determined as a measure for the amount of cells present on the scaffolds. No significant effect of pore size on the amount of DNA present was seen for scaffolds with pore sizes between 250-1000 microm. Light microscopy data showed cells in the center of the scaffolds, more cells were observed in the scaffolds of 425-500 microm and 500-710 microm pore size compared to the ones with 250-425 microm and 710-1000 microm pores.     

In vitro and in vivo characteristics of PCL scaffolds with pore size gradient fabricated by a centrifugation method

Polycaprolactone (PCL) cylindrical scaffolds with gradually increasing pore size along the longitudinal direction were fabricated by a novel centrifugation method to investigate pore size effect on cell and tissue interactions. The scaffold was fabricated by the centrifugation of a cylindrical mold containing fibril-like PCL and the following fibril bonding by heat treatment. The scaffold showed gradually increasing pore size (from approximately 88 to approximately 405 microm) and porosity (from approximately 80% to approximately 94%) along the cylindrical axis by applying the centrifugal speed, 3000 rpm. The scaffold sections were examined for their in vitro cell interactions using different kinds of cells (chondrocytes, osteoblasts, and fibroblasts) and in vivo tissue interactions using a rabbit model (skull bone defects) in terms of scaffold pore sizes. It was observed that different kinds of cells and bone tissue were shown to have different pore size ranges in the scaffold for effective cell growth and tissue regeneration. The scaffold section with 380-405 microm pore size showed better cell growth for chondrocytes and osteoblasts, while the scaffold section with 186-200 microm pore size was better for fibroblasts growth. Also the scaffold section with 290-310 microm pore size showed faster new bone formation than those of other pore sizes. The pore size gradient scaffolds fabricated by the centrifugation method can be a good tool for the systematic studies of the interactions between cells or tissues and scaffolds with different pore size.     

Optimally porous and biomechanically compatible scaffolds for large-area bone regeneration

Large-area or critical-sized bone defects pose a serious challenge in orthopedic surgery, as all current treatment options present with shortcomings. Bone tissue engineering offers a more promising alternative treatment strategy. However, this approach requires mechanically stable scaffolds that support homogenous bone formation throughout the scaffold thickness. Despite advances in scaffold fabrication, current scaffold-based techniques are unable to support uniform, three-dimensional bone regeneration, and are limited to only the scaffold surface in vitro and in vivo. This is mainly because of inadequate scaffold pore sizes (<200?μm) and accessible pore volume, and the associated limited oxygen diffusion and vascular invasion. In this study, we have adopted a method combining microsphere-sintering and porogen-leaching techniques to fabricate scaffolds with an increased accessible pore volume. Of the scaffolds developed, moderately porous poly(85 lactide-co-15 glycolide) (PLGA) microsphere scaffolds were selected as most advantageous, since they retain mechanical strength in the range of human cancellous bone and display a significantly higher accessible pore volume, which is attributed to an increased percentage of larger pores (i.e., size range 200-600?μm). Unlike control scaffolds with a limited pore size and an accessible pore volume, moderately porous scaffolds displayed increased oxygen diffusion, pre-osteoblast cell infiltration, proliferation, and survival throughout the entire scaffold. Furthermore, moderately porous PLGA microsphere scaffolds displayed enhanced and homogenous mineralization in vitro. Since these newly designed moderately porous scaffolds are weight bearing, are fully osteoconductive, and have the ability to support vascularization, they may serve as effective scaffolds for large-area bone defect repair/regeneration. In addition, this study demonstrates the ability to modulate scaffold porosity and, in turn, to develop oxygen tension-controlled matrices that are effective for large-area bone regeneration.     

Biodegradable polymer scaffolds with well-defined interconnected spherical pore network

Scaffolding plays pivotal role in tissue engineering. In this work, a novel processing technique has been developed to create three-dimensional biodegradable polymer scaffolds with well-controlled interconnected spherical pores. Paraffin spheres were fabricated with a dispersion method, and were bonded together through a heat treatment to form a three-dimensional assembly in a mold. Biodegradable polymers such as PLLA and PLGA were dissolved in a solvent and cast onto the paraffin sphere assembly. After dissolving the paraffin, a porous polymer scaffold was formed. The fabrication parameters were studied in relation to the pore shape, interpore connectivity, pore wall morphology, and mechanical properties of the polymer scaffolds. The compressive modulus of the scaffolds decreased with increasing porosity. Longer heat treatment time of the paraffin spheres resulted in larger openings between the pores of the scaffolds. Foams of smaller pore size (100-200 microm) resulted in significantly lower compressive modulus than that of larger pore sizes (250-350 or 420-500 microm). The PLLA foams had a skeletal structure consisting of small platelets, whereas PLGA foams had homogeneous skeletal structure. The new processing technique can tailor the polymer scaffolds for a variety of potential tissue engineering applications because of the well-controlled architecture, interpore connectivity, and mechanical properties.     

Effect of porosity and pore size on microstructures and mechanical properties of poly-epsilon-caprolactone- hydroxyapatite composites

The influence of variant pore-size and porosity on the microstructure and the mechanical properties of poly-epsilon-caprolactone (PCL) and hydroxyapatite (HA) composite were examined for an optimal scaffold in bone tissue engineering. Three various amounts of sodium chloride (NaCl, as porogens) with two distinct particle size ranges (212-355 mum and 355-600 mum) were blended into PCL and HA mixture, followed by a leaching technique to generate PCL-HA scaffolds with various pores and porosity. The porosities of the scaffolds were correlated with the porogen size and concentration. The morphological properties of the resulting scaffolds were assessed by micro-computerized tomography (muCT), scanning electron microscopy (SEM), and energy dispersive X-ray analysis (EDX). Extensive PCL-HA pore interconnections with thinner pore walls were present in scaffolds with higher concentration (4:1 w/w) and larger particulate of porogen used in the fabrication process. Embedding of HA particles in the scaffold resulted in rough surfaces on the composites. Instron actuator testing indicated that the tensile strengths and Young's moduli of scaffolds were influenced by both the porosities and pore sizes of the scaffold. It was apparent that increasing the concentration of porogen compromised the mechanical properties; and a larger porogen particle size led to increased tensile strength but a reduction in Young's modulus. Overall, the data indicated that modification of the concentration and particle size of porogen altered the porous features and mechanical strength of HA-PCL scaffolds. This provided means to manipulate the properties of biocompatible cell-supporting scaffolds for use as bone graft substitutes.     

Pore size effect of collagen scaffolds on cartilage regeneration

Scaffold pore size is an important factor affecting tissue regeneration efficiency. The effect of pore size on cartilage tissue regeneration was compared by using four types of collagen porous scaffolds with different pore sizes. The collagen porous scaffolds were prepared by using pre-prepared ice particulates that had diameters of 150–250, 250–355, 355–425 and 425–500 μm. All the scaffolds had spherical large pores with good interconnectivity and high porosity that facilitated cell seeding and spatial cell distribution. Chondrocytes adhered to the walls of the spherical pores and showed a homogeneous distribution throughout the scaffolds. The in vivo implantation results indicated that the pore size did not exhibit any obvious effect on cell proliferation but exhibited different effects on cartilage regeneration. The collagen porous scaffolds prepared with ice particulates 150–250 μm in size best promoted the expression and production of type II collagen and aggrecan, increasing the formation and the mechanical properties of the cartilage.     

Fabrication of porous ultra-short single-walled carbon nanotube nanocomposite scaffolds for bone tissue engineering

We investigated the fabrication of highly porous scaffolds made of three different materials [poly(propylene fumarate) (PPF) polymer, an ultra-short single-walled carbon nanotube (US-tube) nanocomposite, and a dodecylated US-tube (F-US-tube) nanocomposite] in order to evaluate the effects of material composition and porosity on scaffold pore structure, mechanical properties, and marrow stromal cell culture. All scaffolds were produced by a thermal-crosslinking particulate-leaching technique at specific porogen contents of 75, 80, 85, and 90 vol%. Scanning electron microcopy, microcomputed tomography, and mercury intrusion porosimetry were used to analyze the pore structures of scaffolds. The porogen content was found to dictate the porosity of scaffolds. There was no significant difference in porosity, pore size, and interconnectivity among the different materials for the same porogen fraction. Nearly 100% of the pore volume was interconnected through 20microm or larger connections for all scaffolds. While interconnectivity through larger connections improved with higher porosity, compressive mechanical properties of scaffolds declined at the same time. However, the compressive modulus, offset yield strength, and compressive strength of F-US-tube nanocomposites were higher than or similar to the corresponding properties for the PPF polymer and US-tube nanocomposites for all the porosities examined. As for in vitro osteoconductivity, marrow stromal cells demonstrated equally good cell attachment and proliferation on all scaffolds made of different materials at each porosity. These results indicate that functionalized ultra-short single-walled carbon nanotube nanocomposite scaffolds with tunable porosity and mechanical properties hold great promise for bone tissue engineering applications.     

Resorbable polymeric scaffolds for bone tissue engineering: the influence of their microstructure on the growth of human osteoblast-like MG 63 cells

Degradable three-dimensional porous scaffolds applicable as cell carriers for bone tissue engineering were developed by an innovative solvent casting/particulate leaching technique from poly(L-lactide-co-glycolide) (PLG). Three types of PLG scaffolds were prepared, and these had the same high porosity (83%) but increasing diameter of the pores (180-200 microm, 250-320 microm, and 400-600 microm) and increasing pore interconnectivity. The colonization of the scaffolds with human osteoblast-like MG 63 cells was then studied in vitro in a conventional static cell culture system. The number of cells growing on the scaffolds on days 1 and 7 after seeding was highest in the material with the largest pore diameter, but on day 15, the differences among the scaffolds disappeared. Confocal microscopy revealed that on day 1 after seeding, the cells penetrated to a depth of 490 +/- 100 microm, 720 +/- 170 microm, and 720 +/- 120 microm into the scaffolds of small, medium, and large pore size, respectively. Incorporation of bromodeoxyuridine into newly synthesized DNA and the concentration of vinculin, beta-actin, osteopontin, and osteocalcin in cells on the scaffolds of all pore sizes were similar to the values obtained on standard tissue culture polystyrene, which indicated good biocompatibility of the scaffolds. These results suggest that all scaffolds could serve as good carriers for bone cells, although the quickest colonization with cells was found in the scaffolds with the largest pore diameter from 400 to 600 microm.     

Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin

A new all-aqueous process is described to form three-dimensional porous silk fibroin matrices with control of structural and morphological features. The result of this process are scaffolds with controllable porosity and pore sizes that fully degrade in the presence of proteases, unlike prior methods to generate silk-based biomaterials that required the use of organic solvent treatments to impart control of structure and stability in aqueous environments, with low rates of proteolytic hydrolysis. A mechanism is proposed for this novel process that imparts physical stability via hydrophobic interactions. Adjusting the concentration of silk fibroin in water, and the particle size of granular NaCl used in the process, leads to the control of morphological and functional properties of the scaffolds. The aqueous-derived scaffolds had highly homogeneous and interconnected pores with pore sizes ranging from 470 to 940 microm, depending on the mode of preparation. The scaffolds had porosities >90% and compressive strength and modulus up to 320 +/- 10 and 3330 +/- 500 KPa, respectively, when formed from 10% aqueous solutions of fibroin. The scaffolds fully degraded upon exposure to protease during 21 days, unlike the scaffolds prepared from organic solvent processing. These new silk-based three-dimensional matrices provide useful properties as biomaterial matrices due to the all-aqueous mode of preparation, control of pore size, connectivity of pores, degradability and useful mechanical features. Importantly, this process offers an entirely new window of materials properties when compared with traditional silk fibroin-based materials.     

Fabrication and characterization of poly(propylene fumarate) scaffolds with controlled pore structures using 3-dimensional printing and injection molding

Poly(propylene fumarate) (PPF) is an injectable, biodegradable polymer that has been used for fabricating preformed scaffolds in tissue engineering applications because of in situ crosslinking characteristics. Aiming for understanding the effects of pore structure parameters on bone tissue ingrowth, 3-dimensional (3D) PPF scaffolds with controlled pore architecture have been produced in this study from computer-aided design (CAD) models. We have created original scaffold models with 3 pore sizes (300, 600, and 900 microm) and randomly closed 0%, 10%, 20%, or 30% of total pores from the original models in 3 planes. PPF scaffolds were fabricated by a series steps involving 3D printing of support/build constructs, dissolving build materials, injecting PPF, and dissolving support materials. To investigate the effects of controlled pore size and interconnectivity on scaffolds, we compared the porosities between the models and PPF scaffolds fabricated thereby, examined pore morphologies in surface and cross-section using scanning electron microscopy, and measured permeability using the falling head conductivity test. The thermal properties of the resulting scaffolds as well as uncrosslinked PPF were determined by differential scanning calorimetry and thermogravimetric analysis. Average pore sizes and pore shapes of PPF scaffolds with 600- and 900-microm pores were similar to those of CAD models, but they depended on directions in those with 300-microm pores. Porosity and permeability of PPF scaffolds decreased as the number of closed pores in original models increased, particularly when the pore size was 300 microm as the result of low porosity and pore occlusion. These results show that 3D printing and injection molding technique can be applied to crosslinkable polymers to fabricate 3D porous scaffolds with controlled pore structures, porosity, and permeability using their CAD models.     

The influence of dispersant concentration on the pore morphology of hydroxyapatite ceramics for bone tissue engineering

There is a clinical need for synthetic scaffolds that will promote bone regeneration. Important factors include obtaining an optimal porosity and size of interconnecting windows whilst maintaining scaffold mechanical strength, enabling complete penetration of cells and nutrients throughout the scaffold, preventing the formation of necrotic tissue in the centre of the scaffold. To address this we investigated varying slip deflocculation in order to control the resulting porosity, pore size and interconnecting window size whilst maintaining mechanical strength. Hydroxyapatite (HA) porous ceramics were prepared using a modified slip casting process. Rheological measurements of the HA slips were used to identify deflocculation conditions which resulted in changes in the cell and window sizes of the resulting ceramics. Sintered ceramics were characterised by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Pore and window size distribution was determined by SEM. XRD analysis confirmed that the crystal structure remained HA after the sintering process. SEM showed that HA porous ceramics presented a highly interconnected porous network with average pore sizes ranging from 391+/-39 to 495+/-25 microm. The average window size varied from 73+/-5 to 135+/-7 microm. Pore diameters obtained were controllable in the range 200-500 microm. Window sizes were in the range 30-250 microm. The use of dispersant concentration allows pore and window size to be modified whilst maintaining control over porosity demonstrated by a porosity of 85% for seven different dispersant concentrations. The advantage of this approach allows the correlation between the rheological conditions of the slip and the resultant sintered ceramic properties. In particular, optimising the ceramic strength by controlling the agglomeration during the casting process.     

Indirect solid free form fabrication of local and global porous,biomimetic and composite 3D polymer-ceramic scaffolds

Precise control over scaffold material, porosity, and internal pore architecture is essential for tissue engineering. By coupling solid free form (SFF) manufacturing with conventional sponge scaffold fabrication procedures, we have developed methods for casting scaffolds that contain designed and controlled locally porous and globally porous internal architectures. These methods are compatible with numerous bioresorbable and non-resorbable polymers, ceramics, and biologic materials. Phase separation, emulsion-solvent diffusion, and porogen leaching were used to create poly(L)lactide (PLA) scaffolds containing both computationally designed global pores (500, 600, or 800 microm wide channels) and solvent fashioned local pores (50-100 microm wide voids or 5-10 microm length plates). Globally porous PLA and polyglycolide/PLA discrete composites were made using melt processing. Biphasic scaffolds with mechanically interdigitated PLA and sintered hydroxyapatite regions were fabricated with 500 and 600 microm wide global pores. PLA scaffolds with complex internal architectures that mimicked human trabecular bone were produced. Our indirect fabrication using casting in SFF molds provided enhanced control over scaffold shape, material, porosity and pore architecture, including size, geometry, orientation, branching, and interconnectivity. These scaffolds that contain concurrent local and global pores, discrete material regions, and biomimetic internal architectures may prove valuable for multi-tissue and structural tissue interface engineering.     

Bone formation on the apatite-coated zirconia porous scaffolds within a rabbit calvarial defect

Previously, a strong and bioactive ceramic scaffold consisting of a porous zirconia body coated with apatite double layers (fluorapatite (FA) as an inner layer and hydroxyapatite (HA) as an outer layer) was successfully fabricated. In this contribution, the authors investigate the in vivo performance of the engineered bioceramic scaffolds using a rabbit calvarial defect model. In particular, the porosity and pore size of the scaffolds are varied in order to observe the geometrical effects of the scaffolds on their bone formation behaviors. The scaffolds supported on a zirconia framework can be produced with an extremely high porosity (approximately 84-87%), while retaining excellent compressive strength (approximately 7-8 MPa), which has been unachievable in the case of pure apatite scaffolds (approximately 74% porosity with approximately 2 MPa strength).The experimental groups used in this study include three types of zirconia scaffolds coated with apatite; high porosity (approximately 87%) with large pore size (approximately 500- 700 microm): AZ-HL, high porosity (approximately 84%) with small pore size (approximately 150-200 microm): AZ-HS, and low porosity (approximately 75%) with large pore size (approximately 500-700 microm): AZ-LL, as well as one type of HA porous scaffold: low porosity (approximately 74%) with a large pore size (approximately 500-700 microm) for the purpose of comparison. The scaffolds prepared with dimensions of approximately 10 mm (diameter) x 1.2 mm (thickness) are grafted in rabbit calvaria defects. The histological sections are made at 4 and 12 weeks after surgery and immunohistochemical analyses are performed on the samples.All of the specimens show a good healing response without adverse tissue reactions. Good healing is shown at 4 weeks post-surgery with the ingrowth of new bone into the macropore-channels of the scaffolds. The newly formed bone amounts to approximately 19.9-24.2% of the initial defect area, depending on the scaffold type, but there is no statistical significance between the scaffold groups. However, the defects without the scaffolds (control group) show a significantly lower bone formation ratio (approximately 4.3%). At twelve weeks after surgery, the extent of new bone formation is more pronounced in all of the scaffold groups. All of the scaffold groups show significantly higher bone formation ratios (26.7-46.9%) with respect to the control without the graft. In the comparison between the scaffold groups, those with high porosities (AZ-HL and AZ-HS) exhibit significantly higher bone formation as compared to the scaffold with low porosity (AZ-LL).Based on the present in vivo test performed within a rabbit calvaria defect model, it is concluded that the apatite-coated zirconia scaffolds show good bone forming ability and are considered to be a promising scaffolding material for bone regeneration since they possess a high level of both mechanical and biological properties.     

Repair of mandibular defects using MSCs-seeded biodegradable polyester porous scaffolds

PLLA, PLA-PEG and PLGA porous scaffolds with pore size ranging from 100 to 250 microm and porosity over 85% were fabricated by a solution-casting/salt-leaching method. The porous structure and porosity of the scaffold were mainly dependent on volume fraction and size of the porogens of NaCl particles. The effects of the polymeric materials on the cell culture behavior and bone formation in vitro in their scaffolds were studied. In vitro cell culture in the scaffolds of the three polymers demonstrated that mesenchymal stem cells (MSCs) had a good adhesion and spread. The composite matrixes cultured for several days possessed preliminary functions of tissue-engineering bone, with signs of the calcium knur formation and the expression of osteocalcin and collagen I in mRNA, especially that of PLA-PEG and PLGA. These cell-loaded porous scaffolds showed effective repair of mandibular defect of rabbits in vivo. Contrastive experiments demonstrated that the MSCs/PLGA scaffold owned better ability facilitating for the MSCs proliferation, differentiation and defect repair. These composite scaffolds can be a potential effective tool for treating mandibular and other bone defects.     

Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition.

The aim of this study was to determine the influence of two key scaffold design parameters, void fraction (VF) and pore size, on the attachment, growth, and extracellular matrix deposition by several cell types. Disc-shaped, porous, poly(-lactic acid) (L-PLA) scaffolds were manufactured by the TheriForm solid free-form fabrication process to generate scaffolds with two VF (75% and 90%) and four pore size distributions (< 38, 38-63, 63-106, and 106-150 microm). Microcomputed tomography analysis revealed that the average pore size was generally larger than the NaCl used, while VF was at or near the designated percentage. The response of three cell types-canine dermal fibroblasts (DmFb), vascular smooth muscle cells (VSMC), or microvascular epithelial cells (MVEC)-to variations in architecture during a 4-week culture period were assessed using histology, metabolic activity, and extracellular matrix deposition as comparative metrics. DmFb, VSMC, and MVEC showed uniform seeding on scaffolds with 90% VF for each pore size, in contrast to the corresponding 75% VF scaffolds. DmFb showed the least selectivity for pore sizes. VSMC displayed equivalent cell proliferation and matrix deposition for the three largest pore sizes. MVEC formed disconnected webs of tissue with sparse extracellular matrix at 90% VF and >38 to 150 microm; however, when cultured on scaffolds with pores formed with salt particles of <38 microm, MVEC formed a multilayered lining on the scaffolds surface. Culture data from scaffolds with a 75% VF suggests that the structural features were unsuitable for tissue formation. Hence, there were limits of acceptable scaffold architecture (VF, pore size) that modulated in vitro cellular responses.     

Tissue-engineered growth of bone by marrow cell transplantation using porous calcium metaphosphate matrices

In this study we investigated not only osteoblastic cell proliferation and differentiation on the surface of calcium metaphosphate (CMP) matrices in vitro but also bone formation by ectopic implantation of these cell-matrix constructs in athymic mice in vivo. Interconnected porous CMP matrices with pores 200 microm in size were prepared to use as scaffolds for rat-marrow stromal-cell attachment. Cell-matrix constructs were cultured in vitro, and cell proliferation and ALPase activities were monitored for 56 days. In addition to their being cultured in vitro, cell-matrix constructs were implanted into subcutaneous sites of athymic mice. In vitro these porous CMP matrices supported the proliferation of osteoblastic cells as well as their differentiation, as indicated by high ALPase activity. In vivo the transplanted marrow cells gave rise to bone tissues in the pores of the CMP matrices. A small amount of woven bone formation was detected first at 4 weeks; osteogenesis progressed vigorously with time, and thick lamellar bones that had been remodeled were observed at 12 weeks. These findings demonstrate the potential for using a porous CMP matrix as a biodegradable scaffold ex vivo along with attached marrow-derived mesenchymal cells for transplantation into a site for bone regeneration in vivo.     

Fabrication of three-dimensional polycaprolactone/hydroxyapatite tissue scaffolds and osteoblast-scaffold interactions in vitro

Computer-aided tissue-engineering approach was used to develop a novel precision extrusion deposition (PED) process to directly fabricate Polycaprolactone (PCL) and composite PCL/hydroxyapatite (PCL-HA) tissue scaffolds. The process optimization was carried out to fabricate both PCL and PCL-HA (25% concentration by weight of HA) with a controlled pore size and internal pore structure of the 0 degrees /90 degrees pattern. Two groups of scaffolds having 60% and 70% porosity and with pore sizes of 450 and 750 microm, respectively, were evaluated for their morphology and compressive properties using scanning electron microscopy (SEM) and mechanical testing. Our results suggested that inclusion of HA significantly increased the compressive modulus from 59 to 84 MPa for 60% porous scaffolds and from 30 to 76 MPa for 70% porous scaffolds. In vitro cell-scaffolds interaction study was carried out using primary fetal bovine osteoblasts to assess the feasibility of scaffolds for bone tissue-engineering application. The cell proliferation and differentiation were calculated by Alamar Blue assay and by determining alkaline phosphatase activity. The osteoblasts were able to migrate and proliferate over the cultured time for both PCL as well as PCL-HA scaffolds. Our study demonstrated the viability of the PED process to the fabricate PCL and PCL-HA composite scaffolds having necessary mechanical property, structural integrity, controlled pore size and pore interconnectivity desired for bone tissue engineering.     

The effect of pore geometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4V bone scaffolds

The specific aim of this study was to gain insight into the influence of scaffold pore size, pore shape and permeability on the in vitro proliferation and differentiation of three-dimensional (3-D) human periosteum-derived cell (hPDC) cultures. Selective laser melting (SLM) was used to produce six distinct designed geometries of Ti6Al4V scaffolds in three different pore shapes (triangular, hexagonal and rectangular) and two different pore sizes (500 μm and 1000 μm). All scaffolds were characterized by means of two-dimensional optical microscopy, 3-D microfocus X-ray computed tomography (micro-CT) image analysis, mechanical compression testing and computational fluid dynamical analysis. The results showed that SLM was capable of producing Ti6Al4V scaffolds with a broad range of morphological and mechanical properties. The in vitro study showed that scaffolds with a lower permeability gave rise to a significantly higher number of cells attached to the scaffolds after seeding. Qualitative analysis by means of live/dead staining and scanning electron micrography showed a circular cell growth pattern which was independent of the pore size and shape. This resulted in pore occlusion which was found to be the highest on scaffolds with 500 μm hexagonal pores. Interestingly, pore size but not pore shape was found to significantly influence the growth of hPDC on the scaffolds, whereas the differentiation of hPDC was dependent on both pore shape and pore size. The results showed that, for SLM-produced Ti6Al4V scaffolds with specific morphological and mechanical properties, a functional graded scaffold will contribute to enhanced cell seeding and at the same time can maintain nutrient transport throughout the whole scaffold during in vitro culturing by avoiding pore occlusion.     

Preparation and characterization of a multilayer biomimetic scaffold for bone tissue engineering

In scaffold based bone tissue engineering, both the pore size and the mechanical properties of the scaffold are of great importance. However, an increase in pore size is generally accompanied by a decrease in mechanical properties. In order to achieve both suitable mechanical properties and porosity, a multilayer scaffold is designed to mimic the structure of cancellous bone and cortical bone. A porous nano-hydroxyapatite-chitosan composite scaffold with a multilayer structure is fabricated and encased in a smooth compact chitosan membrane layer to prevent fibrous tissue ingrowth. The exterior tube is shown to have a small pore size (15-40 microm in diameter) for the enhancement of mechanical properties, while the core of the multilayer scaffold has a large pore size (predominantly 70-150 microm in diameter) for nutrition supply and bone formation. Compared with the uniform porous scaffold, the multilayer scaffold with the same size shows an enhanced mechanical strength and larger pore size in the center. More cells are shown to grow into the center of the multilayer scaffold in vitro than into the uniform porous scaffold under the same seeding condition. Finally, the scaffolds are implanted into a rabbit fibula defect to evaluate the osteoconductivity of the scaffold and the efficacy of the scaffold as a barrier to fibrous tissue ingrowth. At 12 weeks post operation, affluent blood vessels and bone formation are found in the center of the scaffold and little fibrous tissue is noted in the defect site.