The effect of PEGT/PBT scaffold architecture on the composition of tissue engineered cartilage

A highly interconnecting and accessible pore network has been suggested as one of a number of prerequisites in the design of scaffolds for tissue engineering. In the present study, two processing techniques, compression-molding/particulate-leaching (CM), and 3D fiber deposition (3DF), were used to develop porous scaffolds from biodegradable poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) co-polymers with varying pore architectures. Three-dimensional micro-computed tomography (microCT) was used to characterize scaffold architectures and scaffolds were seeded with articular chondrocytes to evaluate tissue formation. Scaffold porosity ranged between 75% and 80%. Average pore size of tortuous CM scaffolds (182 microm) was lower than those of organized 3DF scaffolds (525 microm). The weight ratio of glycosaminoglycans (GAG)/DNA, as a measure of cartilage-like tissue formation, did not change after 14 days of culture whereas, following subcutaneous implantation, GAG/DNA increased significantly and was significantly higher in 3DF constructs than in CM constructs, whilst collagen type II was present within both constructs. In conclusion, 3DF PEGT/PBT scaffolds create an environment in vivo that enhances cartilaginous matrix deposition and hold particular promise for treatment of articular cartilage defects.     

Poly(butyl methacrylate-co-methacrylic acid) tissue engineering scaffold with pro-angiogenic potential in vivo

A poly(butyl methacrylate-co-methacrylic acid) (BMA-MAA) scaffold was fabricated by an in situ polymerization solvent casting/particulate leaching technique. It displayed high porosity (85-90%), pore interconnectivity, and a pore size range of 100-650 microm. Compression testing of the scaffolds demonstrated a dependence of the compressive stiffness on several fabrication variables including the ratio of monomer to salt used during the polymerization, the degree of salt fusion, and the choice of alternative comonomers to BMA. Subcutaneous implantation of BMA-MAA scaffolds in mice revealed an increased level of histological angiogenesis in tissue invading the pores of the scaffold compared to a BMA control, consistent with the prediction that methacrylic acid (MAA) containing copolymer beads are angiogenic in a wound healing context. At postoperative day 21, the capillary density in the BMA-MAA scaffolds was 56 +/- 13/mm(2) as compared to 32 +/- 8/mm(2) for the BMA scaffolds. With further investigation, it is expected that this biomaterial capable of eliciting an angiogenic response will have widespread application in tissue engineering.     

Design of porous scaffolds for cartilage tissue engineering using a three-dimensional fiber-deposition technique

In this study, we present and characterize a fiber deposition technique for producing three-dimensional poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT/PBT) block co-polymer scaffolds with a 100% interconnecting pore network for engineering of articular cartilage. The technique allowed us to "design-in" desired scaffold characteristics layer by layer by accurately controlling the deposition of molten co-polymer fibers from a pressure-driven syringe onto a computer controlled x-y-z table. By varying PEGT/PBT composition, porosity and pore geometry, 3D-deposited scaffolds were produced with a range of mechanical properties. The equilibrium modulus and dynamic stiffness ranged between 0.05-2.5 and 0.16-4.33 MPa, respectively, and were similar to native articular cartilage explants (0.27 and 4.10 MPa, respectively). 3D-deposited scaffolds seeded with bovine articular chondrocytes supported a homogeneous cell distribution and subsequent cartilage-like tissue formation following in vitro culture as well as subcutaneous implantation in nude mice. This was demonstrated by the presence of articular cartilage extra cellular matrix constituents (glycosaminoglycan and type II collagen) throughout the interconnected pore volume. Similar results were achieved with respect to the attachment of expanded human articular chondrocytes, resulting in a homogeneous distribution of viable cells after 5 days dynamic seeding. The processing methods and model scaffolds developed in this study provide a useful method to further investigate the effects of scaffold composition and pore architecture on articular cartilage tissue formation.     

Quantitative analysis of interconnectivity of porous biodegradable scaffolds with micro-computed tomography

Pore interconnectivity within scaffolds is an important parameter influencing cell migration and tissue ingrowth needed to promote tissue regeneration. Methods for assessment of interconnectivity are usually qualitative, restricted to two-dimensional images, or are destructive. Microcomputed tomography nondestructively provides three-dimensional (3D) images of intact specimens at high spatial resolutions. We describe an image analysis technique for quantitative assessment of scaffold interconnectivity. Scaffolds were made via a particulate leaching process with 75%, 80%, 85%, and 88% volumetric porogen fractions. Specimens were scanned and resulting 3D, digital images were analyzed with a custom algorithm. A series of virtual, idealized scaffolds were also created for illustration of the algorithm's analysis approach and for its validation. The program calculated accessible void fractions over a range of minimum connection sizes. In real specimens, nearly 100% of the porous volume was connected with outside air for connections greater than or equal to 20 microm in their smallest dimension. In scaffolds made with 75% porogen, the accessible void fraction decreased to 78% if only those connections greater than or equal to 260 microm were considered. The relationship between accessible void fraction and connection size varied as a function of porogen content. The interconnectivity parameter described here may have implications for cell migration and tissue growth into scaffolds.     

Biodegradable composite scaffolds with an interconnected spherical network for bone tissue engineering

Tissue engineering scaffolds are highly engineered structures that accommodate cells, facilitate their expression, and resorb to facilitate regeneration of tissue. A new technique for producing controlled pore shape and pore size interconnectivity offers promise for application as a tissue engineering scaffold. Salt particles were spheroidized in a flame and sintered to provide an interconnecting salt template. The salt template was filled with a carbonated fluorapatite powder and a polylactic polymer to produce a composite scaffold. It was found that a higher pore space is possible with the use of spherical and larger salt particle sizes. This technique can produce scaffolds with good interconnectivity and be suitable for producing pore size graded bodies.     

In vitro assessment of cell penetration into porous hydroxyapatite scaffolds with a central aligned channel

There is a clinical need for synthetic scaffolds that promote bone regeneration. A common problem encountered when using scaffolds in tissue engineering is the rapid formation of tissue on the outer edge of the scaffold whilst the tissue in the centre becomes necrotic. To address this, the scaffold design should improve nutrient and cell transfer to the scaffold centre. In this study, hydroxyapatite scaffolds with random, open porosity (average pore size of 282+/-11microm, average interconnecting window size of 72+/-4microm) were manufactured using a modified slip-casting methodology with a single aligned channel inserted into the centre. By varying the aligned channel diameter, a series of scaffolds with channel diameters ranging from 170 to 421microm were produced. These scaffolds were seeded with human osteosarcoma (HOS TE85) cells and cultured for 8 days. Analysis of cell penetration into the aligned channels revealed that cell coverage increased with increasing channel diameter; from 22+/-3% in the 170microm diameter channel to 38+/-6% coverage in the 421microm channel. Cell penetration into the middle section of the 421microm diameter channel (average cell area coverage 121x10(3)+/-32x10(3)microm(2)) was significantly greater than that observed within the 170microm channel (average cell area coverage 26x10(3)+/-6x10(3)microm(2)). In addition, the data presented demonstrates that the minimum channel (or pore) diameter required for cell penetration into such scaffolds is approximately 80microm. These results will direct the development of scaffolds with aligned macroarchitecture for tissue engineering bone.     

Salt fusion: an approach to improve pore interconnectivity within tissue engineering scaffolds

Macroporous scaffolds composed of biodegradable polymers have found extensive use as three-dimensional substrates either for in vitro cell seeding followed by transplantation, or as conductive substrates for direct implantation in vivo. Methods abound for creation of macroporous scaffolds for tissue engineering, and common methods typically employ a solid porogen within a three-dimensional polymer matrix to create a well-defined pore size, pore structure, and total scaffold porosity. This study describes an approach to impart improved pore interconnectivity to polymer scaffolds for tissue engineering by partially fusing the solid porogen together prior to creation of a continuous polymer matrix. Three dimensional, porous scaffolds of the copolymer 85:15 poly(lactide-co-glycolide) were fabricated via either a solvent casting/particulate leaching process, or a gas foaming/particulate leaching process. Prior to creation of a continuous polymer matrix the NaCl crystals, which serve as the solid porogen, are partially fused via treatment in 95% humidity. Scanning electron micrographs clearly display fused salt crystals and an enhancement in pore interconnectivity in the salt fused scaffolds prepared via both solvent casting and gas foaming, and the extent of pore interconnectivity is enhanced with longer treatment times. Fusion of salt crystal for 24 h increased the radius of curvature of salt crystals, and led to a twofold increase in the compressive modulus of solvent cast scaffolds (total porosity of 97 +/- 1%). Fusion of NaCl crystals prior to gas foaming resulted in a decrease in scaffold compressive modulus from 277 +/- 60k Pa to 187 +/- 30k Pa (total porosity of 94 +/- 1%). The resulting highly interconnected scaffolds have implications for facilitated cell migration, abundant cell-cell interaction, and potentially improved neural and vascular growth within tissue engineering scaffolds.     

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.     

The use of PEGT/PBT as a dermal scaffold for skin tissue engineering

Human skin equivalents (HSEs) were engineered using biodegradable-segmented copolymer PEGT/PBT as a dermal scaffold. As control groups, fibroblast-populated de-epidermized dermis, collagen, fibrin and hybrid PEGT/PBT-collagen matrices were used. Two different approaches were used to generate full-thickness HSE. In the 1-step approach, keratinocytes were seeded onto the fibroblast-populated scaffolds and cultured at the air-liquid (A/L) interface. In the 2-step approach, fully differentiated epidermal sheets were transferred onto fibroblast-populated scaffolds and cultured at the A/L. In a 1-step procedure, keratinocytes migrated into the porous PEGT/PBT scaffold. This was prevented by incorporating fibroblast-populated collagen into the pores of the PEGT/PBT matrix or using the 2-step procedure. Under all experimental conditions, fully differentiated stratified epidermis and basement membrane was formed. Differences in K6, K16, K17, collagen type VII, laminin 5 and nidogen staining were observed. In HSE generated with PEGT/PBT, the expression of these keratins was higher, and the deposition of collagen type VII, laminin 5 and nidogen at the epidermal/matrix junction was retarded compared to control HSEs. Our results illustrate that the copolymer PEGT/PBT is a suitable scaffold for the 2-step procedure, whereas the incorporation of fibroblast-populated collagen or fibrin into the pores of the scaffold is required for the 1-step procedure.     

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.     

Photo-patterning of porous hydrogels for tissue engineering

Since pore size and geometry strongly impact cell behavior and in vivo reaction, the ability to create scaffolds with a wide range of pore geometries that can be tailored to suit a particular cell type addresses a key need in tissue engineering. In this contribution, we describe a novel and simple technique to design porous, degradable poly(2-hydroxyethyl methacrylate) hydrogel scaffolds with well-defined architectures using a unique photolithography process and optimized polymer chemistry. A sphere-template was used to produce a highly uniform, monodisperse porous structure. To create a patterned and porous hydrogel scaffold, a photomask and initiating light were employed. Open, vertical channels ranging in size from 360+/-25 to 730+/-70 microm were patterned into approximately 700 microm thick hydrogels with pore diameters of 62+/-8 or 147+/-15 microm. Collagen type I was immobilized onto the scaffolds to facilitate cell adhesion. To assess the potential of these novel scaffolds for tissue engineering, a skeletal myoblast cell line (C2C12) was seeded onto scaffolds with 147 microm pores and 730 microm diameter channels, and analyzed by histology and digital volumetric imaging. Cell elongation, cell spreading and fibrillar formation were observed on these novel scaffolds. In summary, 3D architectures can be patterned into porous hydrogels in one step to create a wide range of tissue engineering scaffolds that may be tailored for specific applications.     

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.     

Evaluation of chondrogenesis within PEGT: PBT scaffolds with high PEG content

Porous poly(ethylene glycol) terephthalate:poly (butylene terephthalate) (PEGT:PBT) scaffolds with high PEG molecular weight (1000 g/mole) and PEGT content (60%) were fabricated using two different processes-paraffin templating and compression molding-for cartilage engineering applications. This polymer composition has previously been shown to enable chondrocyte adhesion and maintain differentiated phenotype in 2D monolayer culture. The influence of 3D polymer scaffold processing on the formation of cartilaginous tissue was studied by seeding primary immature bovine chondrocytes within cylindrical scaffolds in mixed flask reactors for 3 days, followed by cultivation in culture plates for a total of 10 or 24 days. Tissue-polymer constructs were evaluated morphologically by SEM and histology, and quantitatively for cellularity, total collagen, and glycosaminoglycan content, all of which remained statistically equivalent for each time point tested, irrespective of fabrication method. These data demonstrate that the polymers engineered for this study were able to support chondrogenesis independent of scaffold fabrication process, with the influence of pore architecture lessened by the highly hydrated scaffold microenvironments induced by high PEG content.     

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.     

An initial investigation of photocurable three-dimensional lactic acid based scaffolds in a critical-sized cranial defect

Degradable polymer networks formed by the photoinitiated polymerization of multifunctional monomers have great potential as in situ forming materials, especially for bone tissue engineering. In this study, one specific chemistry was analyzed with respect to bone formation in a critical-sized defect model with and without adsorbed osteoinductive growth factors present. The scaffolds degraded in approximately 8 months and possessed an elastic modulus similar to that of trabecular bone. A porous scaffold fabricated with approximately 80% porosity and pore diameters ranging from 45 to 150 mm was implanted in a critical-sized cranial defect in rats. When implanted alone, the scaffolds were filled primarily with fibrous tissue after 9 weeks with only mild inflammation at the defect site. When the scaffolds released osteoinductive growth factors, statistically more bone filled the scaffold. For instance, 65.8+/-9.4% (n=5) of the defects were filled with radiopaque tissue in the osteoinductive releasing scaffolds, whereas only 24.2+/-7.4% (n=5) of the defects were filled in the untreated defects 9 weeks after implantation. These results illustrate not only the benefits of delivering osteoinductive factors when developing synthetic bone grafts, but the potential of these materials for supporting the infiltration and development of bone in large defects.     

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.     

新型可降解聚合物聚乙二醇对苯二甲酸酯/聚对苯二甲酸丁二醇酯的细胞相容性研究

目的：研究新型可降解聚合物聚乙二醇对苯二甲酸酯／聚对苯二甲酸丁二醇酯(PEGT／PBT)的人脐静脉内皮细胞(HUVEC)相容性，对其在组织工程血管中的应用进行探讨。方法：对PEGT／PBT进行细胞毒性评价。观察并检测HUVEC在PEGB／PBT、Ⅰ型胶原改性(Col-)的PEGT／PBT、纤维连接蛋白改性(Fn-)的PEGT／PBT上的粘附和增殖，对细胞在粘附过程中的粘着斑蛋白进行免疫荧光染色观察。结果：PEGT／PBT细胞毒性不大于1级，能支持脐静脉内皮细胞的粘附和增殖。纤维连接蛋白和Ⅰ型胶原处理可促进HLIVEC在PEGT／PBT膜上的增殖，而且纤维连接蛋白可增加HUVEC在PEGT／PBT上20min、2h的粘附率，并促进细胞形成局部粘附结构。结论：新型可降解聚合物PEGT／PBT具有良好的血管细胞相容性，对其在组织工程血管中的应用值得进一步开发研究。     

Smooth muscle cell adhesion in surface-modified three-dimensional copolymer scaffolds prepared from co-continuous blends

In this article, tissue-engineering scaffolds were fabricated from P(epsilon-CL-co-D,L-LA)-PEG-P(epsilon-CL-co-D,L-LA) copolymers using co-continuous blends with polystyrene as the porogen phase. By means of static annealing and following extraction of the porogen phase, pore sizes (channel widths) in the range of 15-350 microm were obtained. Smooth muscle cells were seeded in three-dimensional fibronectin-modified scaffolds of two different pore sizes. Considerably enhanced cell seeding efficiency was found for scaffolds with larger pore sizes, indicating the importance of this parameter to promote effective cell intrusion into bulk materials. Compressive moduli ranged from 2.3 +/- 0.3 to 67 +/- 15 MPa and decreased with increasing pore size. The reverse trend was found for scaffold permeability (kappa), which ranged from 8.5 x 10(-16) to 6.7 x 10(-11) m(2). This was comparable with permeabilities previously reported for scaffolds with higher pore sizes and void volumes, but more irregular pore morphologies. Taken together, the results obtained in this study motivate further investigation for possible future applications in tissue engineering.     

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.     

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.