Vascular endothelial growth factor-bound stents: application of in situ capture technology of circulating endothelial progenitor cells in porcine coronary model.

OBJECTIVES: We evaluated the in vivo performance of a newly devised vascular endothelial growth factor (VEGF)-bound stent in a porcine coronary model. BACKGROUND: An anti-CD34 antibody-bound stent, which captures endothelial progenitor cells (EPCs) to accelerate tissue formation, did not reduce intimal hyperplasia. By targeting the VEGF receptor, which is expressed on endothelial-lineage cells, we developed VEGF-bound stents that may enable selective capture of EPCs followed by rapid endothelialization. METHODS: Metallic stents were first coated with poly-(ethylene-co-vinyl alcohol), and then chemically bound with either VEGF or anti-CD34 antibody. These stents were placed in porcine coronary arteries for up to 14 days. Stent surface was evaluated by immunohistochemistry and by scanning electron microscope (SEM). RESULTS: After 2-day stenting with VEGF-bound stents, small populations of KDR (VEGF receptor-2)-positive cells adhered to the stent struts. After 7- and 14-day stenting, struts were fully covered with newly regenerated tissue. SEM images showed that the uniform tissue formed on struts was morphologically similar to native endothelium and was continuously connected with adjacent native endothelium. On the other hand, for the anti-CD34 antibody-bound stents, stent struts were rapidly covered by newly generated tissue that consisted of multicellular aggregates. CONCLUSIONS: Compared with anti-CD34 antibody-bound stents, VEGF-bound stents provide highly selective capture of EPCs, followed by rapid formation of intact endothelium tissue at an early period of stenting. These results suggest that VEGF-bound stents could represent a promising therapeutic option for cardiovascular stenting, although further long-term follow-up experiment with double-blinded fashion is needed prior to clinical application.

[Development of elastameric sealant designed for arterial field].

The development of a reliable surgical sealant specific for arterial tissues has been long awaited. In this article, first the "ideal" adhesion mechanism formulated from biomechanical concept is proposed for ensured hemostasis in dissected arteries with pulsatile flow. An urethane prepolymer prepared along the design criteria is viscous liquid. Due to its high water absorbility and high reactivity with water, the sealant applied to vascular tissues becomes an elastomer within several minutes. When the sealant was applied to dissected canine abdominal arteries with 3 stay sutures, followed by declamping 5 minutes, neither bleeding nor detrimental effect on tissue morphogenesis was observed. This sealant is being ready to the market.

[Development of hemostatic sealant for arterial anastomosis; clinical application].

For the purpose of examining the clinical applicability of a newly developed surgical sealant, animal experiments were performed, and clinical trial was followed. In animal experiments, several animal models, including carotid artery anastomosis model and coronary artery bypass grafting model were undertaken. In each model, complete hemostasis of the anastomoses using four simple interrupted sutures, was obtained. In addition, elastomeric property of the sealant prevented thinning of the arterial wall. The clinical trial performed in patients with thoracic aortic surgery showed significantly better hemostasis even under heparinized condition. Based on these excellent results, clinical usage of the sealant was approved.

Arterial shear stress augments the differentiation of endothelial progenitor cells adhered to VEGF-bound surfaces.

Our ongoing studies show that vascular endothelial cell growth factor (VEGF)-bound surfaces selectively capture endothelial progenitor cells (EPCs) in vitro and in vivo, and that surface-bound VEGF stimulates intracellular signal transduction pathways over prolonged culture periods, resulting in inductive differentiation of EPCs. In this article, we investigated whether simulated arterial shear stress augments the differentiation of EPCs adhered to a VEGF-bound surface. Human peripheral blood-derived mononuclear cells adhered to a VEGF-bound surface were exposed to 1 day of shear stress (15 dynes/cm(2), corresponding to shear load in arteries). Shear stress suppressed the expression of mRNAs encoding CD34 and CD133, which are markers for EPCs, and augmented the expression of mRNAs encoding CD31 and von Willebrand factor (vWF) as well as vWF protein, which are markers for endothelial cells (ECs). Shear stress enhanced expression of ephrinB2 mRNA, a marker for arterial ECs, but did not significantly change expression of EphB4 mRNA, a marker for venous ECs. Focused protein array analysis showed that mechanotransduction by shear stress activated the p38 and MAPK pathways in EPCs. Thus, arterial shear stress, in concert with surface-bound VEGF, augments the differentiation of EPCs. These results strongly support previous observation of rapid differentiation of EPCs captured on VEGF-bound stents in a porcine model.

Randomized clinical trial of an elastomeric sealant for hemostasis in thoracic aortic surgery.

OBJECTIVES: This study aimed to demonstrate the efficacy and safety of a newly developed elastomeric sealant, which does not require any blood coagulation system to exert its effect, during thoracic aortic surgery. METHODS: This is a multicenter, randomized study conducted in six hospitals in Japan. A total of 81 patients undergoing replacement surgery of a thoracic aortic aneurysm using cardiopulmonary bypass were randomized with a ratio of 2-:1 for those patients designated to receive the sealant (Group S, 54 patients) or those without the usage of the sealant (Group C, 27 patients). The primary endpoints were bleeding from each anastomosis at two time points: (1) immediately before applying protamine and (2) 15 min after applying protamine. The patients were followed for 6 months. RESULTS: The number of anastomoses checked for bleeding was 196 in Group S and 117 in Group C. Before protamine sulfate administration, complete hemostasis was obtained in 155 anastomoses (79%) in Group S compared to 45 anastomoses (38%) in Group C (p < 0.001). Fifteen minutes after the administration of protamine sulfate infusion, bleeding stopped completely in 173 anastomoses (88%) in Group S and in 71 anastomoses (61%, p < 0.001) in Group C. Between the two groups, there were no marked differences in the patient background or in the incidence of major adverse events. CONCLUSIONS: The sealant is effective in achieving hemostasis, even under fully heparinized conditions. The novel sealant is safe and effective in thoracic aortic surgery, one of the most demanding surgical situations for hemostasis.

Simultaneous processing of fibril formation and cross-linking improves mechanical properties of collagen.

In vitro "simultaneous processing" was investigated in which fibril formation of collagen and cross-linking occur simultaneously in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) as a cross-linking reagent. Fibril formation in simultaneous processing was monitored using turbidity. The EDC in simultaneous processing increased T(1/2) (time required for half of the plateau value in turbidity) and decreased the degree of the fibril formation dose dependently. The reduced fibril formation rate (T(1/2) > 60 s) suggests the introduction of intrafibrillar cross-linking during fibril formation. The collagen gels prepared using simultaneous processing had a compressive modulus that was 6-fold higher than that using sequential processing, which is an advantage of simultaneous processing. Atomic force microscopy images acquired under water on the wet gels demonstrated that the simultaneous processing provided a unique double-network structure: intrafibrillarly cross-linked collagen fibrils among which nonfibrous collagens act as interfibrillar cross-linkages.

Microelastic gradient gelatinous gels to induce cellular mechanotaxis.

The understanding and realization of directional cell movement towards a harder region of a cell culture substrate surface, so-called mechanotaxis, might provide a solid basis for a functional artificial extracellular matrix, enabling manipulation and elucidation of cell motility. The photolithographic surface microelasticity patterning method was developed for fabricating a cell-adhesive hydrogel with a microelasticity gradient (MEG) surface using photocurable styrenated gelatin to investigate the condition of surface elasticity to induce mechanotaxis as a basis for such substrate-elasticity-dependent control of cell motility. Patterned MEG gels consisting of different absolute surface elasticities and elasticity jumps were prepared. Surface elasticity and its two-dimensional distribution were characterized by microindentation tests using atomic force microscopy (AFM). From analyses of trajectories of 3T3 cell movement on each prepared MEG gel, two critical criteria of the elasticity jump and the absolute elasticity to induce mechanotaxis were identified: (1) a high elasticity ratio between the hard region and the soft one, and (2) elasticity of the softer region to provide medium motility. Design of these conditions was found to be necessary for fabricating an artificial extracellular matrix to control or manipulate cell motility.

In situ harvesting of adhered target cells using thermoresponsive substrate under a microscope: principle and instrumentation.

A novel technique and instrumented device were developed to harvest target cells from multicellular mixture of different cell types under a microscope. The principle of the technique is that cells cultured on a thermoresponsive-substance-coated dish were detached by a region-specific cooling device and simultaneously harvested using a micropipette, both of which were assembled in an inverted microscope. Thermoresponsive coating consists of the mixture of poly(N-isopropylacrylamide) (PNIPAAm) and PNIPAAm-grafted gelatin. The former non-cell-adhesive polymer dissolves below at 32.1 degrees C in water and precipitates over that temperature (called lower critical solution temperature, LCST), and the latter cell-adhesive polymer has LCST of 34.1 degrees C. The appropriate mixing ratio of these thermoresponsive polymers exhibited high cell adhesion at physiological temperature and complete cell detachment at room temperature. A device developed as to cool at only a tiny area of the bottom of the dish, beneath which a cell that was targeted under a microscope, was assembled in a microscope. It was demonstrated that single cell or two cells that adhered to each other was detached from the surface and harvested by a micropipette within approximately 30s.

Development of biodegradable scaffolds based on patient-specific arterial configuration.

Biodegradable scaffolds are of great value in tissue engineering. We have developed a method for fabricating patient-specific vascular scaffolds from a biocompatible and biodegradable polymer, poly(L-lactide-co-epsilon-caprolactone). This method's usefulness is due to flexibility in the choice of materials and vascular configurations. Here, we present a way to fabricate scaffolds of human carotid artery by combining processes of rapid prototyping, lost wax, dip coating, selective dissolution, and salt leaching. The result was the successful development of porous biodegradable scaffolds, with mechanical strength covering the range of human blood vessels (1-3 MPa). Human umbilical vein endothelial cells were also cultured on the scaffolds and their biocompatibility was confirmed by cell growth. The Young's modulus of scaffolds could be controlled by changing polymer concentration and porosity. The wall thickness of the tubular scaffold was also controllable by adjusting polymer concentration and pull-up velocity during dip coating. We believe that this fabrication technique can be applied to patient-specific regeneration of blood vessels.

Matrix metalloproteinases and their inhibitors in the foreign body reaction on biomaterials.

Matrix metalloproteinases (MMPs) can degrade structural components within the extracellular matrix and at the cellular surface producing changes in cellular behavior (i.e., adhesion and migration) and subsequent pathological responses (i.e., the foreign body reaction and wound healing). We continue to study the foreign body reaction that occurs following biomaterial implantation by investigating secretory responses of biomaterial-adherent macrophages and foreign body giant cells (FBGCs) as directed by material surface chemistry and further this research by determining whether secreted MMPs play a role in macrophage adhesion and fusion. We have identified numerous MMPs and their tissue inhibitors (TIMPs) in in vitro cell-culture supernatants using antibody arrays and quantified select MMP/TIMPs with ELISAs. MMP-9 concentrations were significantly greater than both TIMP-1 and TIMP-2 on all materials. The ratios of MMP-9/TIMP-1 and MMP-9/TIMP-2 increased with time because of an increase in MMP-9 concentrations over time, while the TIMP concentrations remained constant. Total MMP-9 concentrations in the supernatants were comparable on all materials at each timepoint, while TIMP-1 and TIMP-2 concentrations tended to be greater on hydrophilic/anionic surfaces. Analysis of the MMP/TIMP quantities produced per cell revealed that the hydrophilic/neutral surfaces, which inhibited macrophage adhesion, activated the adherent macrophages/FBGCs to produce a greater quantity of MMP-9, TIMP-1, and TIMP-2 per cell. Pharmacological inhibition of MMP-1,-8,-13, and -18 reduced macrophage fusion without affecting adhesion, while inhibitors of MMP-2,-3,-9, and -12 did not affect adhesion or fusion. These findings demonstrate that material surface chemistry does modulate macrophage/FBGC-derived MMP/TIMP secretion and implicates MMP involvement in macrophage fusion.

The effect of gradually graded shear stress on the morphological integrity of a huvec-seeded compliant small-diameter vascular graft.

The premature endothelialization of tissue-engineered grafts had often induced cellular detachment at an early period of implantation in arterial circulation, resulting in occlusion at an early period of implantation. This study was aimed to determine whether gradually increased shear stress applied ex vivo improves cell retention and tissue morphological integrity including cell shape and alignment, actin fiber alignment and expression of vascular endothelial (VE) cadherin. Tissue-engineered grafts used for this study were human umbilical vein endothelial cell (HUVEC)-seeded compliant small-diameter grafts made of poly(L-lactide-co-epsilon-caprolactone) fiber meshes fabricated by electrospinning. The shear stresses applied to grafts, generated using a custom-designed mock circulatory apparatus, were 3.2, 8.7 and 19.6 dyn/cm(2). The grafts completely monolayered prior to shear stress exposure exhibited a polygonal cobblestone morphology with randomly distributed actin fibers and VE cadherin at the continuous peripheral region of adjacent cells. The 24-h-loading of high shear stresses (8.7 and 19.6 dyn/cm(2)) equivalent to those of the arterial circulatory system resulted in severe cellular damage resulting in the complete loss of cells. However, a gradually increased graded exposure from a low (3.2 dyn/cm(2)) to a high shear stress (19.6 dyn/cm(2)) resulted in a markedly reduced cell detachment, a highly elongated cell shape, and orientation or alignment of both cells and actin fibers, which were parallel to the direction of flow. Although VE-cadherin expression was not detected yet, a higher degree of tissue integrity was achieved, which may greatly improve the performance particularly at an early period of implantation.

Mechanical loading-dependence of mRNA expressions of extracellular matrices of chondrocytes inoculated into elastomeric microporous poly(L-lactide-co-epsilon-caprolactone) scaffold.

The temporal response of young rabbit chondrocyte metabolism (including biosynthesis of extracellular matrix macromolecules such as collagen and aggrecan, both of which are essential components of normal cartilage tissue, and their messenger ribonucleic acid (mRNA) expression) in microporous elastomeric scaffolds made of poly(L-lactide-co-epsilon-caprolactone) subjected to different compressive regimes (loading frequency, loading duration per cycle, loading period, and continuous or intermittent compression) were studied over a 6-day culture period at 10% of compressive strain. A continuous dynamic compression improved the production of sulfated glycosaminoglycan (S-GAG), most of which was released into the culture medium upon loading. High mRNA expression of type II collagen was exhibited at a frequency of 0.1 Hz. Little frequency dependency was observed for aggrecan. An intermittent loading (24-h cycle of loading and unloading) or short loading and unloading duration per cycle-compression regime maintained high levels of mRNA expression. This strongly suggests that well-controlled mechanical conditioning regimes may control the gene expression of key metabolic substances relevant to functional cartilage tissue while the degree of release of these substances into the culture medium is minimized.

Shape-engineered fibroblasts: cell elasticity and actin cytoskeletal features characterized by fluorescence and atomic force microscopy.

The regulation of cell shape, which determines cell behaviors including adhesion, spreading, migration, and proliferation in an engineered artificial extracellular milieu, is an important task in tissue engineering and in development of functional biomaterials. To deepen the understandings of shape-dependent cell mechanics, the cell elasticity and structural features of the actin cytoskeleton (CSK) were characterized for shape-engineered fibroblasts; round and spindle-shaped cells cultured on photolithographically microprocessed surfaces, employing the cellular microindentation tests and fluorescence observation of actin CSK by the combination of atomic force microscopy (AFM) and fluorescence microscopy (FM). The relationships among cell elasticity, the structural features of actin CSK, and engineered cell shape were analyzed and compared with those of control cells that had been cultured on nonprocessed surfaces (termed naturally extended cells). Results showed that the spindle-shaped cells with sparse or no apical stress fibers (ASFs) exhibited similar stiffness to that of the naturally extended cells with dense ASFs. The elasticity of spindle-shaped cells was affected only slightly by the stress fiber (SF) density, which is in marked contrast to the significant correlation shown between cell elasticity and SF density in naturally extended cells. This result implies that the elasticity of regionally restricted adhesion-surface-induced shape-engineered cells, particularly of highly elongated cells, is affected predominantly by cell shape rather than by structural features of SFs.

Shape-engineered vascular endothelial cells: nitric oxide production, cell elasticity, and actin cytoskeletal features.

Single cell shape determines cellular functions. Therefore, control of cell shape is of considerable importance for the tissue engineering field. This study was designed to assess the effect of surface-induced shaping of vascular endothelial cells (ECs) on the intracellular nitric oxide (NO) production level, the cell elasticity, and cytoskeletal (CSK) features on shape-engineered ECs (round, 90, 120 microm diameter; spindle-shaped, 20, 30, 40 microm width) prepared on a photolithographically microprocessed surface. Intracellular NO production was measured using a microscopic spectrometer with diaminofluorescein diacetate probe. Cell elasticity and actin CSK features were analyzed through microindentation measurement and fluorescence observations with fluorescence and atomic force microscopy. Results showed that spindle-shaped cells exhibited lower NO production, higher cell stiffness, and denser actin stress fibers than the round and nonrestrictedly cultured control cells. Relations between cell shape with NO production, cell elasticity, and actin CSK features were discussed.

Proteomic analysis and quantification of cytokines and chemokines from biomaterial surface-adherent macrophages and foreign body giant cells.

Implantation of biomaterial devices results in the well-known foreign body reaction consisting of monocytes, macrophages, and foreign body giant cells (FBGCs) at the material/tissue interface. We continue to address the hypothesis that material surface chemistry modulates the phenotypic expression of these cells. Utilizing our human monocyte culture system, we have used surface-modified polymers displaying hydrophobic, hydrophilic, and/or ionic chemistries to determine the cytokines/chemokines released from biomaterial-adherent macrophages/FBGCs. This study broadens our approach by using proteomic analysis to identify important factors expressed by these cells and further quantifies these molecules with ELISAs. Proteomic profiles changed over time suggesting that the adherent macrophages underwent a phenotypic switch. Macrophage/FBGC-derived proinflammatory cytokines, IL-1beta and IL-6, decreased with time, while the anti-inflammatory cytokine, IL-10, gradually increased with time. Resolution of the inflammatory response was also demonstrated by a decrease in chemoattractant IL-8 and MIP-1beta production with time. Material-dependent macrophage/FBGC activation was analyzed using cytokine/chemokine production and cellular adhesion. Monocyte/macrophage adhesion was similar on all surfaces, except for the hydrophilic/neutral surfaces that showed a significant decrease in cellular density and minimal FBGC formation. Normalizing the ELISA data based on the adherent cell population provided cytokine/chemokine concentrations produced per cell. This analysis showed that although there were fewer cells on the hydrophilic/neutral surface, these adherent cells were further activated to produce significantly greater amounts of each cytokine/chemokine tested than the other surfaces. This study clearly presents evidence that material surface chemistry can differentially affect monocyte/macrophage/FBGC adhesion and cytokine/chemokine profiles derived from activated macrophages/FBGCs adherent to biomaterial surfaces.

Elastomeric surgical sealant for hemostasis of cardiovascular anastomosis under full heparinization.

PURPOSE: We developed a novel surgical sealant, a viscous diisocyanated prepolymer, applicable to arterial hemostasis. The purpose of this study is to evaluate hemostatic effect of this surgical sealant under heparinized conditions. METHODS: The effectiveness of this sealant was verified by applying it to the end-to-end anastomosis of canine carotid arteries. Five mongrel dogs were used. After a complete heparinization, the carotid arteries were clamped, divided, and end-to-end anastomoses were performed with four simple interrupted sutures. The sealant was coated on the anastomosis. After 5 min the clamps were removed and the hemostatic effect was evaluated. Three dogs were immediately subjected to macroscopic evaluation. Two dogs were subjected to angiography after 3 months and 16 months, respectively. RESULTS: No bleeding occurred in any of the anastomoses immediately after the removal of the clamp. Macroscopic finding revealed no leakage of the sealant into the lumen. Carotid angiography revealed patent anastomoses without stenosis. CONCLUSION: A novel surgical sealant exhibited rapid and potent hemostatic effect on a moisturized tissue under full heparinization.

Photo-iniferter-based thermoresponsive block copolymers composed of poly(ethylene glycol) and poly(N-isopropylacrylamide) and chondrocyte immobilization.

A series of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM)-poly(ethylene glycol) (PEG) block copolymers with various PNIPAM contents and copolymer architectures, such as linear, four-armed and eight-armed configurations, were prepared by iniferter-based photopolymerization of dithiocarbamylated PEGs (DC-PEGs) under ultraviolet (UV)-light irradiation. The increase in monomer/DC-PEG feed ratio resulted in an increase in both the molecular weight and PNIPAM content of copolymers. The measurement of the optical transmittances of aqueous solutions of PNIPAM-PEG block copolymers determined the lower critical solution temperatures (LCSTs) of block copolymers, which ranged from 31.3 to 34.0 degrees C. LCST decreased with increasing block length of PNIPAM and with the formation of a branched architecture. Rabbit chondrocytes were immobilized and cultured in a three-dimensional (3D) gel composed of PNIPAM-PEG block copolymer at 37 degrees C. Gels prepared from copolymers with higher PNIPAM contents at higher concentrations appeared to exhibit a minimal decrease in both cell number and cell viability during a 7-day culture. Cell viability dependencies on material and formulation parameters and the potential use of PNIPAM-PEG block copolymers as an in situ formable scaffold for an engineered cartilage tissue were discussed.

Mechanical responses of a compliant electrospun poly(L-lactide-co-epsilon-caprolactone) small-diameter vascular graft.

To design a "mechano-active" small-diameter artificial vascular graft, a tubular scaffold made of elastomeric poly(L-lactide-co-epsilon-caprolactone) fabrics at different wall thicknesses was fabricated using an electrospinning (ELSP) technique. The wall thickness of the fabricated tube (inner diameter; approximately 2.3-2.5 mm and wall thickness; 50-340 microm) increased proportionally with ELSP time. The wall thickness dependence of mechanical responses including intraluminal pressure-induced inflation was determined under static and dynamic flow conditions. From the compliance-related parameters (stiffness parameter and diameter compliance) measured under static condition, the smaller the wall thickness, the more compliant the tube. Under dynamic flow condition (1 Hz, maximal/minimal pressure of 90 mmHg/45 mmHg) produced by a custom-designed arterial circulatory system, strain, defined as the relative increase in diameter per pulse, increased with the decrease in wall thickness, which approached that of a native artery. Thus, a mechano-active scaffold that pulsates synchronously by responding to pulsatile flow was prepared using elastomeric PLCL as a base material and an ELSP technique.

Encapsulation of chondrocytes in photopolymerizable styrenated gelatin for cartilage tissue engineering.

We have developed a photopolymerizable styrenated gelatin that can cross-link through polymerization induced by irradiation with visible light. The purpose of this study was to investigate the feasibility of using photopolymerizable styrenated gelatin as a cell carrier in chondrocyte transplantation. As visible light activates camphorquinone added as a photoinitiator, free radicals induce the polymerization of the gelatin macromer; the styrenated gelatin then becomes cross-linked. Rabbit articular chondrocytes were cultured in styrenated gelatin hydrogels and also in collagen gels as a control. After being cultured in the gels, the cells were collected from both gels and counted. Reverse transcriptase-polymerase chain reaction, histological examination, and quantification of the synthesized glycosaminoglycan were performed. On average, 26% of the embedded cells were collected from the gelatin hydrogel immediately after the crosslinking reaction. The surviving chondrocytes expressed the mRNA of type II collagen and aggrecan core protein and produced a cartilaginous matrix throughout the gelatin after 3 weeks. A slightly insufficient accumulation of the matrix was found in the internal region of the gelatin hydrogels, suggesting that less permeability for nutrients due to the high concentration and closely packed structure resulted in less cell viability. Although some limitations became evident, these results indicate that it may be possible to use photopolymerizable styrenated gelatin as a cell carrier in chondrocyte transplantation.

New technique of seeding chondrocytes into microporous poly(L-lactide-co-epsilon-caprolactone) sponge by cyclic compression force-induced suction.

The initial requirement for a functional engineered cartilage tissue is the effective and reproducible seeding of chondrocytes into the interior of microporous scaffolds. High seeding efficiency, high cell viability, uniform cell distribution, and short operation time are also essential. We devised a new technique of seeding rabbit chondrocytes into microporous poly(L-lactide-co-epsilon-caprolactone) (PLCL) (porosity, 71- 80%; wall thickness, 2 and 6 mm) sponges under compression force-induced suction using a custom designed loading apparatus. Cell distribution and cell viability were determined using confocal laser scanning microscopy with fluorescent dye-staining techniques. Factors that affect the quality of a cell seeded construct were studied, namely, the porosity and thickness of sponges and suction cycles. Under 1 cycle of suction, an increase in porosity promoted cell seeding efficiency (CSE; defined as the percentage of the number of cells in the sponges relative to the initial number of cells seeded), cell viability (at 1 day post seeding), and a relatively uniform cell distribution, whereas thick sponges exhibited an inhomogeneous cell distribution irrespective of incubation time. Multiple cycles of suction of 5 and 10 at 0.1 Hz significantly improved the CSE, whereas high cell viability was maintained and even spatial cell distribution was achieved in 1 week. This study revealed that our newly developed cell seeding technique with multiple cycles of suction is a promising approach to inoculating cells into microporous sponges with high CSE, high cell viability, and homogeneous cell distribution.