Formation of granulation tissue in subcutaneously implanted sponges in rats. A comparison between granulation tissue developed in viscose cellulose sponges (Visella) and in polyvinyl alcohol sponges (Ivalon).

A comparison was made between the granulation tissue formation in two different synthetic sponge types. Visella and Ivalon, of different sizes. The granulation tissue formed in the two sponge types did not differ qualitatively, and had the character of wound tissue and inflammatory tissue in man. The rate of tissue formation in the Visella sponges was faster and the tissue was more homogenous than in the Ivalon sponges. Fourteen-day-old Visella implants of either size contained more granulation tissue than Ivalon sponges, probably owing to the smaller pore size of the former material. This may also account for the more frequent occurrence of giant cells in the Visella implants. In contrast to the Visella sponges, the trabeculae of the Ivalon polymer showed calcification and positive staining properties with histological staining procedures, and deformation was frequent among the Ivalon implants. Thin sponges of either type closed in about 21 days, thick ones after about 42 days of implantation. Calculated per 2 cm3 of implant, thin sponges produced more tissue after 14 days of implantation than thick noes. It is concluded that the Visella sponge type is best suitable for this experimental model of inflammation.     

Preparation of collagen/gelatin sponge scaffold for sustained release of bFGF

Artificial dermis (AD) has been used to regenerate dermis-like tissues in the treatment of full-thickness skin defects, but it takes 2 or 3 weeks to complete dermal regeneration. Our previous study demonstrated that injection of basic fibroblast growth factor (bFGF)-impregnated gelatin microspheres (MS) into the AD accelerates the regeneration of dermis-like tissue. However, injection of gelatin MS before clinical use is complicated and time consuming. This study investigated a new scaffold, in which collagen and gelatin are integrated, and which is capable of sustained bFGF release. We produced collagen/gelatin sponges with a gelatin concentration of 0wt%, 10wt%, 30wt%, and 50wt%. The mean pore size in each sponge decreased with the gelatin concentration. In an in vitro study, proliferation of fibroblasts in each sponge was not significantly different over 7 days of culture. As for in vivo sustained release of bFGF, a radioisotope study demonstrated that retention of bFGF in gelatin 10wt% and 30wt% sponges was significantly larger than that in gelatin 0wt% sponge. The collagen/gelatin sponges were grafted on full-thickness skin defects created on a rabbit ear, and we evaluated regeneration of dermis-like tissue by measuring the amount of hemoglobin and size of dermis-like tissue on histological sections. Seven days after implantation, the amount of hemoglobin in dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in control and gelatin 50wt% sponge. Twenty-eight days after implantation, the area of dermis-like tissue in gelatin 10wt% sponge was significantly larger than those in the other specimens. We conclude that the collagen sponge integrated with 10wt% gelatin has the most potential for sustained release of bFGF and that the combination of collagen/gelatin 10wt% sponge and bFGF is a promising therapeutic modality for the treatment of full-thickness skin defects.     

Quantitative in-vivo studies on angiogenesis in a rat sponge model.

A method for quantitative in-vivo studies on angiogenesis is described in this article. It is based on subcutaneous implantation of sterile polyester sponges in the rat and subsequent measurement of blood flow in the implants as they become vascularized. The blood flow in an implant was measured in terms of per cent 133Xe-saline clearance 6 min after the radio-isotope was injected into the sponge via a cannula attached to it. Since originally the sponge contained no blood vessels, the development of blood flow would represent a neovascularization. Histological examination of implants removed at fixed time intervals confirmed that the sponges were initially encapsulated by granulation tissue and gradually infiltrated by host blood vessels. Under standard conditions, the 133Xe clearance from sponges 16 days post-implantation approached the clearance obtained in normal skin. The new blood vessels in the sponges were reactive to vasodilator prostaglandin-E2 and vasoconstrictor noradrenaline applied topically. Furthermore, we have shown that local administration of endothelial cell growth supplement accelerated angiogenesis while protamine delayed its onset. Thus the model offers a new means for objective, continuous and reproducible studies on the controlling mechanisms of angiogenesis.     

Quantitative in-vivo studies on angiogenesis in a rat sponge model

A method for quantitative in-vivo studies on angiogenesis is described in this article. It is based on subcutaneous implantation of sterile polyester sponges in the rat and subsequent measurement of blood flow in the implants as they become vascularized. The blood flow in an implant was measured in terms of per cent 133Xe-saline clearance 6 min after the radio-isotope was injected into the sponge via a cannula attached to it. Since originally the sponge contained no blood vessels, the development of blood flow would represent a neovascularization. Histological examination of implants removed at fixed time intervals confirmed that the sponges were initially encapsulated by granulation tissue and gradually infiltrated by host blood vessels. Under standard conditions, the 133Xe clearance from sponges 16 days post-implantation approached the clearance obtained in normal skin. The new blood vessels in the sponges were reactive to vasodilator prostaglandin-E2 and vasoconstrictor noradrenaline applied topically. Furthermore, we have shown that local administration of endothelial cell growth supplement accelerated angiogenesis while protamine delayed its onset. Thus the model offers a new means for objective, continuous and reproducible studies on the controlling mechanisms of angiogenesis.     

In vitro culture characteristics of corneal epithelial, endothelial, and keratocyte cells in a native collagen matrix

The objective of this investigation was to demonstrate the effectiveness of a tissue-engineered collagen sponge as a substrate for the culture of human corneal cells. To that end, human kerotocyte, epithelial, and endothelial cells were cultured separately on collagen sponges composed of native fibrillar collagen with a pore size of approximately 0.1 mm. Co-culture experiments were also performed (epithelial/endothelial and epithelial/keratocyte cultures). Proliferation of keratocytes and matrix production was assessed. The morphology of the epithelial and endothelial cell cultures was characterized by histology and scanning electron microscopy. Keratocytes cultured on collagen sponges exhibited increased matrix synthesis over time as well as proliferation and repopulation of the matrix. Epithelial and endothelial cells showed the ability to migrate over the collagen sponge. The thickness of the epithelial layer was influenced by soluble factors produced by endothelial cells. The morphology of the bottom layer of epithelial cells was influenced by the presence of keratocytes in the culture. These studies indicate that human corneal cells exhibit normal cell phenotype when cultured individually on an engineered collagen sponge matrix and co-culture of different cell types in the cornea can influence cell behavior.     

Gelatin-based resorbable sponge as a carrier matrix for human mesenchymal stem cells in cartilage regeneration therapy

Adult mesenchymal stem cells (MSCs), found in the bone marrow, have the potential to differentiate into multiple connective tissue types, including cartilage. In this study, we examined the potential of a porous gelatin sponge, Gelfoam, for use as a delivery vehicle for MSCs in cartilage regeneration therapy. Adult human MSCs (hMSCs) were seeded throughout the gelatin sponge after a 2-h incubation period. When cultured for 21 days in vitro in a defined medium supplemented with 10 ng/mL of TGF-beta 3, hMSC/Gelfoam constructs produced a cartilage-like extracellular matrix containing sulfated glycosaminoglycans (s-GAGs) and type-II collagen, as evident upon histologic evaluation. Constructs loaded with a cell suspension of 12 x 10(6) cells/mL produced an extracellular matrix containing 21 microg of s-GAG/microg of DNA after 21 days of culture. This production was more efficient than constructs loaded at higher or lower cell densities, indicating that the initial seeding density influences the ability of cells to produce extracellular matrix. When implanted in an osteochondral defect in the rabbit femoral condyle, Gelfoam cylinders were observed to be very biocompatible, with no evidence of immune response or lymphocytic infiltration at the site. Based on these observations we conclude that Gelfoam resorbable gelatin sponge is a promising candidate as a carrier matrix for MSC-based cartilage regeneration therapies.     

Combination of BMP-2-releasing gelatin/β-TCP sponges with autologous bone marrow for bone regeneration of X-ray-irradiated rabbit ulnar defects

The objective of this study is to evaluate the feasibility of gelatin sponges incorporating β-tricalcium phosphate (β-TCP) granules (gelatin/β-TCP sponges) to enhance bone regeneration at a segmental ulnar defect of rabbits with X-ray irradiation. After X-ray irradiation of the ulnar bone, segmental critical-sized defects of 20-mm length were created, and bone morphogenetic protein-2 (BMP-2)-releasing gelatin/β-TCP sponges with or without autologous bone marrow were applied to the defects to evaluate bone regeneration. Both gelatin/β-TCP sponges containing autologous bone marrow and BMP-2-releasing sponges enhanced bone regeneration at the ulna defect to a significantly greater extent than the empty sponges (control). However, in the X-ray-irradiated bone, the bone regeneration either by autologous bone marrow or BMP-2 was inhibited. When combined with autologous bone marrow, the BMP-2 exhibited significantly high osteoinductivity, irrespective of the X-ray irradiation. The bone mineral content at the ulna defect was similar to that of the intact bone. It is concluded that the combination of bone marrow with the BMP-2-releasing gelatin/β-TCP sponge is a promising technique to induce bone regeneration at segmental bone defects after X-ray irradiation.     

Adipose tissue engineering by human adipose-derived stromal cells

Tissue engineering has emerged as a promising alternative approach to current clinical treatments for restoration of soft tissue defects. The purpose of this study was to investigate adipose tissue formation in vitro and in vivo by using human adipose-derived stromal cells (ADSCs) utilizing a gelatin sponge (Gelform) as a scaffold. Adipogenic potentials of human ADSCs were demonstrated by Oil-O-red staining and cellular morphology. After seeding human ADSCs in a density of 3 x 10(6) cells/ml on three-dimensional gelatin sponges, tissue-engineered constructs were exposed to adipogenic differentiation medium for in vitro studies and implanted in the backs of severe combined immunodeficient (SCID) mice for in vivo adipose regeneration. Adipogenesis of ADSC-seeded gelatin sponges was confirmed by Oil-O-red staining after 4 weeks of in vitro incubation. The optical density of the elution from Oil-O-red staining of adipogenic constructs is significantly higher than that of the control group (p < 0.05, n = 4). With short-term in vitro differentiation, adipogenic constructs turned into fat tissue 4 weeks after in vivo implantation, confirmed by biochemical and immunohistochemical examination. No adipogenic-morphological change or fat formation was observed in in vitro or in vivo studies when ADSCs were exposed to a control medium without adipogenic stimulation. These results indicate that engineered adipose tissue can be achieved using human ADSCs and biocompatible and degradable gelatin sponges.     

Enhanced osteoinduction by controlled release of bone morphogenetic protein-2 from biodegradable sponge composed of gelatin and beta-tricalcium phosphate

Biodegradable gelatin sponges at different contents of beta-tricalcium phosphate (beta-TCP) were fabricated to allow bone morphogenetic protein (BMP)-2 to incorporate into them. The in vivo osteoinduction activity of the sponges incorporating BMP-2 was investigated, while their in vivo profile of BMP-2 release was evaluated. The sponges prepared had an interconnected pore structure with an average pore size of 200 microm, irrespective of the beta-TCP content. The in vivo release test revealed that BMP-2 was released in vivo at a similar time profile, irrespective of the beta-TCP content. The in vivo time period of BMP-2 retention was longer than 28 days. When the osteoinduction activity of gelatin or gelatin-beta-TCP sponges incorporating BMP-2 was studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, homogeneous bone formation was histologically observed throughout the sponges, although the extent of bone formation was higher in the sponges with the lower contents of beta-TCP. On the other hand, the level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges decreased with an increase in the content of beta-TCP. The gelatin sponge exhibited significantly higher osteoinduction activity than that of any gelatin-beta-TCP sponge, although every sponge with or without beta-TCP showed a similar in vivo profile of BMP-2 release. In addition, the in vitro collagenase digestion experiments revealed that the gelatin-beta-TCP sponge collapsed easier than the gelatin sponge without beta-TCP incorporation. These results suggest that the maintenance of the intrasponge space necessary for the osteoinduction is one factor contributing to the osteoinduction extent of BMP-2-incorporating sponges.     

Regulation of local host-mediated anti-tumor mechanisms by cytokines: direct and indirect effects on leukocyte recruitment and angiogenesis.

The regulation of tumor growth by cytokine-induced alterations in host effector cell recruitment and activation is intimately associated with leukocyte adhesion and angiogenic modulation. In the present study, we have developed a novel tumor model to investigate this complex series of events in response to cytokine administration. Gelatin sponges containing recombinant human basic fibroblast growth factor (rhFGFb) and B16F10 melanoma cells were implanted onto the serosal surface of the left lateral hepatic lobe in syngeneic C57BL/6 mice. The tumor model was characterized by progressive tumor growth initially localized within the sponge and the subsequent development of peritoneal carcinomatosis. Microscopic examination of the sponge matrix revealed well developed tumor-associated vascular structures and areas of endothelial cell activation as evidenced by leukocyte margination. Treatment of mice 3 days after sponge implantation with a therapeutic regimen consisting of pulse recombinant human interleukin-2 (rhIL-2) combined with recombinant murine interleukin-12 (rmIL-12) resulted in a marked hepatic mononuclear infiltrate and inhibition of tumor growth. In contrast to the control group, sponges from mice treated with rhIL-2/rmIL-12 demonstrated an overall lack of cellularity and vascular structure. The regimen of rhIL-2 in combination with rmIL-12 was equally effective against gelatin sponge implants of rhFGFb/B16F10 melanoma in SCID mice treated with anti-asialo-GM1 in the absence of a mononuclear infiltration, suggesting that T, B, and/or NK cells were not the principal mediators of the anti-tumor response in this tumor model. The absence of vascularity within the sponge after treatment suggests that a potential mechanism of rhIL-2/rmIL-12 anti-tumor activity is the inhibition of neovascular growth associated with the establishment of tumor lesions. This potential mechanism could be dissociated from the known activities of these two cytokines to induce the recruitment and activation of host effector cells. Moreover, this model provides a unique opportunity to study the cellular and molecular mechanism(s) underlying both tumor angiogenesis and leukocyte recruitment to metastatic lesions.     

A novel tissue-engineered trachea with a mechanical behavior similar to native trachea

A novel tissue-engineered trachea was developed with appropriate mechanical behavior and substantial regeneration of tracheal cartilage. We designed hollow bellows scaffold as a framework of a tissue-engineered trachea and demonstrated a reliable method for three-dimensional (3D) printing of monolithic bellows scaffold. We also functionalized gelatin sponge to allow sustained release of TGF-β1 for stimulating tracheal cartilage regeneration and confirmed that functionalized gelatin sponge induces cartilaginous tissue formation in vitro. A tissue-engineered trachea was then created by assembling chondrocytes-seeded functionalized gelatin sponges into the grooves of bellows scaffold and it showed very similar mechanical behavior to that of native trachea along with substantial regeneration of tracheal cartilage in vivo. The tissue-engineered trachea developed here represents a novel concept of tracheal substitute with appropriate mechanical behavior similar to native trachea for use in reconstruction of tracheal stenosis.     

In vitro and in vivo characterization of an impervious polyester arterial prosthesis: the Gelseal Triaxial® graft

Over the years, textile polyester arterial prostheses have acquired an excellent reputation for easy handling and good healing characteristics. Until recently, the main drawback in using them was the need for preclotting. This, however, is no longer true. Nonporous polyester grafts which have been coated with an impervious bioerodible layer during manufacture are now commercially available. The Gelseal Triaxial® prosthesis is one of this new generation of grafts. It is manufactured by impregnating a Triaxial® prosthesis with a gelatin coating. An in vivo and in vitro evaluation of this new device has found that its water permeability is almost zero. It has good handling and conformability characteristics, and its bursting strength is slightly greater than that of the uncoated prosthesis due, no doubt, to the presence of the gel. The rates of degradation of the gelatin coating have proven to be rapid under both in vitro and in vivo conditions. In fact, only a few traces of the gel were found remaining on the graft after 2 wk in the canine thoracic aorta. In addition, this study has demonstrated that the use of a bioerodible gelatin coating, with its ability to promote cellular regeneration, is a feasible approach with which to achieve earlier and more complete biological healing.     

Vascularization into a porous sponge by sustained release of basic fibroblast growth factor

Vascularization into a poly(vinyl alcohol) (PVA) sponge was investigated using basic fibroblast growth factor (bFGF). This growth factor was impregnated into biodegradable gelatin microspheres for its sustained release and then the bFGF-containing microspheres or free bFGF were incorporated into PVA sponges. Following subcutaneous implantation into the back of mice, the bFGF-containing gelatin microspheres induced vascularization in and around the sponge to a significantly greater extent than that of free bFGF from 3 days after implantation. Significant ingrowth of fibrous tissue into the sponge was also observed when bFGF-containing microspheres were added to the sponge in contrast to free bFGF. Tissue ingrowth occurred into the deeper portion of the sponge over time while it accompanied formation of new capillaries. Empty gelatin microspheres had no effect on vascularization and the level of fibrous tissue ingrowth into the sponge was similar to that of the control group. It was concluded that incorporation of gelatin microspheres containing bFGF into the PVA sponge was effective in prevascularization of the sponge pores.     

Vascularization into a porous sponge by sustained release of basic fibroblast growth factor

Vascularization into a poly(vinyl alcohol) (PVA) sponge was investigated using basic fibroblast growth factor (bFGF). This growth factor was impregnated into biodegradable gelatin microspheres for its sustained release and then the bFGF-containing microspheres or free bFGF were incorporated into PVA sponges. Following subcutaneous implantation into the back of mice, the bFGF-containing gelatin microspheres induced vascularization in and around the sponge to a significantly greater extent than that of free bFGF from 3 days after implantation. Significant ingrowth of fibrous tissue into the sponge was also observed when bFGF-containing microspheres were added to the sponge in contrast to free bFGF. Tissue ingrowth occurred into the deeper portion of the sponge over time while it accompanied formation of new capillaries. Empty gelatin microspheres had no effect on vascularization and the level of fibrous tissue ingrowth into the sponge was similar to that of the control group. It was concluded that incorporation of gelatin microspheres containing bFGF into the PVA sponge was effective in prevascularization of the sponge pores.     

Effects of high molecular weight hyaluronan on chondrocytes cultured within a resorbable gelatin sponge

Freshly isolated bovine articular chondrocytes were seeded into a resorbable gelatin sponge and cultured in the absence or presence of extrinsic high molecular weight hyaluronan (HA) for up to 1 month. The gelatin sponge could be uniformly and reproducibly loaded with chondrocytes. Immunostaining demonstrated that accumulation of pericellular HA increased in the presence of extrinsic HA. However, this approach could not differentiate between extrinsic and endogenous HA. More chondrocytes were retained within the loaded sponges in the presence of HA. Both cell number and matrix synthesis were increased in the presence of high molecular weight HA throughout the time course. Proteoglycan synthesis per cell increased by 22-fold in the presence of HA at 500 microg/mL. Our model demonstrates that HA can be used as a tool not only to expand freshly isolated chondrocyte numbers but also to increase matrix synthesis and deposition within a resorbable gelatin sponge. Autologous chondrocytes for tissue engineering are always in short supply, so this could be a useful tool with which to increase the retention of cells seeded into other types of scaffold matrices before implanting them into a cartilage defect.     

Biocompatibility and biodegradation of cross-linked gelatin/hyaluronic acid sponge in rat subcutaneous tissue

A gelatin/hyaluronic acid (GH) sponge has been fabricated by freeze-drying and cross-linking. The GH sponge was insoluble when cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The morphologies of sponges were investigated using a field emission scanning electron microscope. The porosity of the GH sponge increased with hyaluronic acid content. The GH sponge was biodegradable, as evidenced by implantation in Wistar rat subcutaneous connective tissue. Fibroblasts infiltrated into the sponge matrix, and regenerated collagen in the matrix to a level of 25% by 15 days after surgery. The GH73 sponge induced an acute inflammatory response compared with the GH91 sponge. This inflammatory response could have been stimulated by the presence of hyaluronic acid up to Day 10, as it decreased afterwards. The C-reactive protein of blood samples also indicated the same result. The blood tests and histological results show that GH sponges have good biocompatibility and low antigenicity for tissue engineering scaffolds.     

Biocompatibility and biodegradation of cross-linked gelatin/hyaluronic acid sponge in rat subcutaneous tissue

A gelatin/hyaluronic acid (GH) sponge has been fabricated by freeze-drying and cross-linking. The GH sponge was insoluble when cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The morphologies of sponges were investigated using a field emission scanning electron microscope. The porosity of the GH sponge increased with hyaluronic acid content. The GH sponge was biodegradable, as evidenced by implantation in Wistar rat subcutaneous connective tissue. Fibroblasts infiltrated into the sponge matrix, and regenerated collagen in the matrix to a level of 25% by 15 days after surgery. The GH73 sponge induced an acute inflammatory response compared with the GH91 sponge. This inflammatory response could have been stimulated by the presence of hyaluronic acid up to Day 10, as it decreased afterwards. The C-reactive protein of blood samples also indicated the same result. The blood tests and histological results show that GH sponges have good biocompatibility and low antigenicity for tissue engineering scaffolds.