Fibroblast growth on a porous collagen sponge containing hyaluronic acid and fibronectin

We have previously shown that the presence of fibronectin (FN) and/or hyaluronic acid (HA) in a 3-dimensional type I collagen sponge enhances wound healing in vivo. In the present study the same material was used as a support for growth of fibroblasts in vitro. Using radiochemical techniques, scanning electron and light microscopy, the properties of fibroblasts cultured on the collagen sponge or on the sponge containing HA or FN have been compared with cultures grown on plastic dishes. Fibroblast replication and collagen synthesis were higher on plastic than on the collagen sponge. In the presence of HA or FN the entire thickness of the sponge was infiltrated by fibroblasts which rapidly replicated. The presence of HA or FN increased synthesis of collagen which was largely deposited around cells.     

Characterization of a cultured dermal substitute composed of a spongy matrix of hyaluronic acid and collagen combined with fibroblasts

 The authors have developed an allogeneic cultured dermal substitute (CDS) through cultivation of fibroblasts on a two-layered spongy matrix of hyaluronic acid (HA) and atelocollagen (Col). The Col spongy layer is essential for attachment and proliferation of fibroblasts on the two-layered spongy matrix. The HA spongy layer is necessary for maintaining the moisture environment on the wound surface. The optimal weight ratio of HA/Col is determined by considering the following characteristics: mechanical properties for handling, cell viability after thawing, potency of vascular endothelial growth factor (VEGF) release after thawing, efficacy of wound healing, and manufacturing cost. This study is designed to investigate the physical properties for handling, the growth behavior of fibroblasts on the spongy matrix, and the quantitative analysis of VEGF released from fibroblasts in the fresh or cryopreserved CDS. The results of this study suggest that a CDS composed of Col spongy matrix alone has the highest potency in regard to the release of VEGF. However, taking into account the manufacturing cost, coupled with the potency of VEGF release, a two-layered sponge of HA and Col with a weight ratio of 5/2 is very promising for commercial application. Received: October 4, 2002 / Accepted: March 20, 2003     

Preparation of porous collagen/hyaluronic acid hybrid scaffolds for biomimetic functionalization through biochemical binding affinity

This study demonstrated the feasibility of introducing an avidin-biotin system to three-dimensional and highly porous scaffolds for the purpose of designing scaffolds that have binding affinity with bioactive molecules for various biomimetic modifications. Porous hybrid scaffolds composed of collagen and hyaluronic acid (HA) were prepared by a novel overrun process. The overrun-processed scaffolds showed a uniform dual-pore structure because of the injection of gas bubbles and ice recrystallization during the fabrication process and had a higher porosity than scaffolds prepared by a conventional freeze-drying method. The mechanical strength and biodegradation kinetics were controlled by the method of preparation and the composition of collagen/HA. Collagen/HA scaffolds did not show any significant adverse effects on cell viability even after 10 days of incubation. The fibroblasts cultured in the overrun-processed scaffolds were widely distributed and had proliferated on the surfaces of the macropores in the scaffolds, whereas the cells that were seeded in the freeze-dried scaffolds had attached mainly on the dense surface of the scaffolds. As the collagen content in the scaffolds increased, the cellular ingrowth into the inner pores of the scaffolds decreased because of the high affinity between the collagen and the cells. Measurements obtained via confocal microscopy revealed that the porous collagen/HA scaffolds could be functionalized with the biotin by incorporating avidin. Therefore, the present biotinylation approach may allow the incorporation of various bioactive molecules (DNA, growth factors, drug, peptide, etc) into the three-dimensional porous scaffolds.     

Spongy matrix of hyaluronic acid and collagen as a cultured dermal substitute: evaluation in an animal test

 The authors have developed a two-layered spongy matrix composed of hyaluronic acid (HA) and atelo-collagen (Col) as a cultured dermal substitute (CDS). This spongy matrix was applied to a full-thickness skin defect on the dorsum of Sprague-Dawley rats, and the wound conditions were observed over a period of 2 weeks. A piece of two-layered spongy matrix was applied to the wound, over which a commercially available polyurethane film dressing was applied. Both the macroscopic and the histological evaluations indicate that the two-layered spongy matrix is able to prepare a highly vascularized granulation tissue at an early stage. These findings suggest that this type of spongy matrix would be useful as a CDS. Received: October 4, 2002 / Accepted: January 23, 2003 Correspondence to:Y. Kuroyanagi     

Production of morphology-controllable porous hyaluronic acid particles using a spray-drying method

Hyaluronic acid (HA) porous particles with controllable porosity and pore size, ranging from 100 to 300 nm, were successfully prepared using a colloidal templating and spray-drying method. HA powder and polystyrene latex (PSL) particles, which were used as the precursor and templating agent, respectively, were mixed in aqueous solution and spray-dried using a two-fluid nozzle system to produce HA and PSL composite particles. Water was evaporated during spray-drying using heated air with a temperature of 120 °C. This simple process was completed within several seconds. The prepared particles were collected and washed with an organic solvent to dissolve the PSL templating agent. The porosity and pore size of the resulting particles were easily controlled by changing the initial mass ratio of precursor to templating agent, i.e., HA to PSL, and by altering the size of the PSL template particles.     

Adhesion and proliferation of human dermal fibroblasts on collagen matrix

The purpose of this study was to evaluate adhesion and growth of human dermal fibroblasts on a 0.150 mm-thick matrix of reconstituted collagen isolated from horse tendon. Collagen was extracted and polymerized according to the standard procedures (Opocrin, Corlo, Modena, Italy). By light microscopy, the bottom surface of the matrix appeared linear and compact, whereas the superficial one was indented and less homogeneous. By scanning electron microscopy, the collagen fibrils had different diameters and the great majority of them was oriented parallel to the surface of the gel. By transmission electron microscopy, collagen fibrils showed the typical banding. Human dermal fibroblasts were seeded on the collagen matrix, previously equilibrated in growth medium. Fibroblast proliferation stopped in the second week and was always significantly lower than that of the same cell strain seeded on plastic and cultured in parallel. By light microscopy, after six days culture, cells formed a confluent multilayer on the surface of the gel. By scanning and transmission electron microscopy, fibroblasts appeared flat and adherent to the matrix. Contacts of cells among themselves and with the collagen fibrils were observed. Fibroblasts never moved into the collagen gel. In conclusion, human dermal fibroblasts can be grown in a three-dimensional matrix made by horse tendon that, on the other hand, seems to condition their proliferation rate.     

Chemical sintering generates uniform porous hyaluronic acid hydrogels

The implantation of scaffolds for tissue repair has achieved only limited success due primarily to the inability to achieve vascularization within the construct. Many strategies have therefore moved to incorporate pores into the scaffolds to encourage rapid cellular infiltration and subsequent vascular ingrowth. We utilized an efficient chemical sintering technique to create a uniform network of polymethyl methacrylate (PMMA) microspheres for porous hyaluronic acid hydrogel formation. The porous hydrogels generated from chemical sintering possessed pore uniformity and interconnectivity comparable to the commonly used non- and heat sintering techniques. Moreover, a similar cell response to the porous hydrogels generated from each sintering approach was observed in cell viability, spreading and proliferation in vitro, as well as cellular invasion in vivo. We propose chemical sintering of PMMA microspheres using a dilute acetone solution as an alternative method to generate porous hyaluronic acid hydrogels since it requires equal or 10-fold less processing time as the currently used non-sintering or heat sintering technique, respectively.     

Development of a cultured dermal substitute composed of a spongy matrix of hyaluronic acid and atelo-collagen combined with fibroblasts: fundamental evaluation

We have developed an allogeneic cultured dermal substitute (CDS) by cultivating fibroblasts on a 2-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Col). The HA sponge was designed to have a honeycomb structure with many holes (0.5 mm diameter) separated by a distance of 4 mm. Part of the Col sponge was able to penetrate into these holes, and the resulting anchoring structure allows binding of a HA spongy layer with a Col spongy layer. The preparation of the CDS consists of two steps: (i) attachment of cells to the Col surface of the hydrated 2-layered spongy matrix and (ii) proliferation of cells on this sponge immersed in culture medium. The aim of the present study was to assess properties of fresh and cryopreserved CDS. Fibroblasts seeded on the Col surface of the 2-layered spongy matrix attached, proliferated and released vascular endothelial growth factor (VEGF) and fibronectin. The amount of VEGF released from cryopreserved CDS after thawing slowly in an incubator at 37°C and re-cultivation for 1 week was about 300 pg/ml. After thawing quickly in a water bath at 37°C and re-cultivation for 1 week, the amount of VEGF released was about 600 pg/ml. These findings indicate that the cryopreserved CDS maintained its ability to release a significant amount of VEGF. Retention of the therapeutic properties of CDS after cryopreservation is important for clinical use.     

Development of a cultured dermal substitute composed of a spongy matrix of hyaluronic acid and atelo-collagen combined with fibroblasts: fundamental evaluation

We have developed an allogeneic cultured dermal substitute (CDS) by cultivating fibroblasts on a 2-layered spongy matrix of hyaluronic acid (HA) and atelo-collagen (Col). The HA sponge was designed to have a honeycomb structure with many holes (0.5 mm diameter) separated by a distance of 4 mm. Part of the Col sponge was able to penetrate into these holes, and the resulting anchoring structure allows binding of a HA spongy layer with a Col spongy layer. The preparation of the CDS consists of two steps: (i) attachment of cells to the Col surface of the hydrated 2-layered spongy matrix and (ii) proliferation of cells on this sponge immersed in culture medium. The aim of the present study was to assess properties of fresh and cryopreserved CDS. Fibroblasts seeded on the Col surface of the 2-layered spongy matrix attached, proliferated and released vascular endothelial growth factor (VEGF) and fibronectin. The amount of VEGF released from cryopreserved CDS after thawing slowly in an incubator at 37 degrees C and re-cultivation for 1 week was about 300 pg/ml. After thawing quickly in a water bath at 37 degrees C and re-cultivation for 1 week, the amount of VEGF released was about 600 pg/ml. These findings indicate that the cryopreserved CDS maintained its ability to release a significant amount of VEGF. Retention of the therapeutic properties of CDS after cryopreservation is important for clinical use.     

Development of cultured dermal substitute composed of hyaluronic acid and collagen spongy sheet containing fibroblasts and epidermal growth factor

The present study aimed to develop a two-layered cultured dermal substitute (CDS). The upper layer is a hyaluronic acid (HA) and collagen (Col) spongy sheet with or without epidermal growth factor (EGF). The lower layer is a HA spongy sheet and Col gel containing fibroblasts. The CDS is prepared in serum-free medium, followed by placing on the wound surface. Corresponding to clinical application, CDS was incubated in serum-free medium for a period of 1, 3 or 5?days, followed by placing onto the air and culture medium interface (wound surface model), and culture for 6?days using conventional culture medium supplemented with serum. Metabolic activity and cytokine production were considerably higher in EGF-incorporating CDS, as compared with EGF-free CDS. Metabolic activity of EGF-incorporating CDS was maintained for a period of 3?days, but decreased slightly after 5?days. EGF-incorporating CDS is able to effectively stimulate fibroblasts within CDS to release increased amounts of vascular endothelial growth factor and hepatocyte growth factor, which are essential for wound healing. CDS is promising for wound therapy, because there is no risk of cellular damage caused by cryopreservation, thawing and rinsing processes. The critical issue is how to reduce the cellular damage during a prolonged period of incubation in serum-free medium. EGF-incorporating CDS can be used after a period of 3–5?days incubation in serum-free medium. This period is sufficient for transport of CDS from manufacturing facilities to hospitals.     

Development of an artificial dermis composed of hyaluronic acid and collagen

This study aimed to investigate the efficacy of an artificial dermis composed of hyaluronic acid (HA) and collagen (Col) with or without epidermal growth factor (EGF), both in in vitro and in vivo. The cross-linked high molecular weight HA spongy sheet was prepared by freeze-drying. The spongy sheet was immersed in a mixed solution of high molecular weight HA, low molecular weight HA, and heat-denatured Col, and then lyophilized to obtain a two-layered spongy sheet. Cross-linking among Col molecules was induced by ultraviolet irradiation to prepare the artificial dermis (Type I). In a similar manner, a two-layered artificial dermis containing EGF (Type II) was prepared using a similar mixed solution containing EGF. The in vitro experiments demonstrated that EGF released from the Type II artificial dermis stimulates fibroblasts to produce increased amounts of vascular endothelial growth factor and hepatocyte growth factor. The therapeutic efficacy of artificial dermis was evaluated in animal tests using Sprague Dawley (SD) rats. The dorsal skin of the SD rat was shaved and then exposed to boiling water for 3?s to induce a deep dermal burn. The necrotic tissue was then excised 3?days later. Each artificial dermis was applied to the skin defect for 7?days and assessed for its ability to generate a wound bed. The in vivo experiments demonstrated that the Type II artificial dermis promotes angiogenesis to a greater extent at an early stage (within 3?days), and also suppresses the inflammatory reaction more successfully compared with the Type I artificial dermis. In further animal tests, an autologous skin graft was performed by excising a piece of skin from the abdominal region and then grafting it onto the wound bed prepared using each artificial dermis for 7?days. Although the Type II artificial dermis had the highest potential to promote angiogenesis, in this animal study, each artificial dermis induced excellent wound bed formation acceptable for autologous skin grafting.     

Non-viral DNA delivery from porous hyaluronic acid hydrogels in mice

The lack of vascularization within tissue-engineered constructs remains the primary cause of construct failure following implantation. Porous constructs have been successful in allowing for vessel infiltration without requiring extensive matrix degradation. We hypothesized that the rate and maturity of infiltrating vessels could be enhanced by complementing the open pore structure with the added delivery of DNA encoding for angiogenic growth factors. Both 100 and 60 μm porous and non-porous hyaluronic acid hydrogels loaded with pro-angiogenic (pVEGF) or reporter (pGFPluc) plasmid nanoparticles were used to study the effects of pore size and DNA delivery on angiogenesis in a mouse subcutaneous implant model. GFP-expressing transfected cells were found inside all control hydrogels over the course of the study, although transfection levels peaked by week 3 for 100 and 60 μm porous hydrogels. Transfection in non-porous hydrogels continued to increase over time corresponding with continued surface degradation. pVEGF transfection levels were not high enough to enhance angiogenesis by increasing vessel density, maturity, or size, although by 6 weeks for all pore size hydrogels more hydrogel implants were positive for vascularization when pVEGF polyplexes were incorporated compared to control hydrogels. Pore size was found to be the dominant factor in determining the angiogenic response with 60 μm porous hydrogels having more vessels/area present than 100 μm porous hydrogels at the initial onset of angiogenesis at 3 weeks. The results of this study show promise for the use of polyplex loaded porous hydrogels to transfect infiltrating cells in vivo and guide tissue regeneration and repair.     

Natural polyelectrolyte films based on layer-by layer deposition of collagen and hyaluronic acid

The aim of the present work was to assemble extracellular matrix components into polyelectrolyte multilayers using the layer-by-layer deposition method. The films are constructed with type-I collagen and hyaluronic acid. The construction exhibits the general features observed during polyelectrolyte multilayer buildup: alternate positive and negative values of the zeta potential of the film during its construction and regular increase of the film thickness with the number, n, of deposition step. This increase is shown to be linear with n. As expected for a linearly growing film, the confocal microscopy shows that when the film is brought in contact with a collagen solution, collagen does not diffuse into the film but interacts only with its outer layer. However, the films are not constituted of homogeneously distributed polyanion/polycation complexes as it is usually observed, but they are formed of fibers as imaged by AFM. The typical width of these fibers increases with the number of deposition steps. Finally, it is found that chondrosarcoma cells spread well and synthesize extracellular matrix components only on the collagen ending films, whereas no cellular matrix was found for HA ending ones. Such architectures may be further functionalized by inclusion of active drugs, peptides, proteins..., and could be used as tunable biomaterial interfaces.     

Immobilization of hyaluronic acid on plasma-sprayed porous titanium coatings for improving biological properties

In the present study, hyaluronic acid (HyA) was covalently immobilized onto titanium coatings to improve their biological properties. Diffuse reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were employed to characterize the HyA-modified titanium coating. HyA-modified titanium coatings possess better cell-material interaction, and human mesenchymal stem cells present good adhesive morphologies on the surface of TC-AAH. The results of subsequent cellular evaluation showed that the immobilization of HyA on titanium coatings could improve hMSC attachment, proliferation, and differentiation. In vivo evaluation of implants in rabbit femur condyle defect model showed improvements of early osseointegration and bone-to-implant contact of TC-AAH. In conclusion, immobilization of HyA could improve biological properties of titanium coatings.     

Behavior of isolated rat oval cells in porous collagen scaffold

The oval cell is regarded as a compensatory cell in liver injury, and is thought to be equivalent to liver stem/progenitor cells. Oval cells were induced by the 2-AAF/CCl(4) dietary method in Fischer 344 rats, and were isolated from excised liver by the collagenase perfusion, enzyme treatment, and cell cloning method. Transmission electron microscopy observation and double immunofluorescence methods were used to characterize the cells. We have developed an in vitro system consisting of three-dimensional collagen and hormonal and cytokine factors. Over 3 weeks, albumin secretion and urea detoxification rate were estimated to assess the biological function of the oval cells cultured in a scaffold. Oval cells cultured in the scaffold demonstrated higher biological functions than did those in a two-dimensional tissue culture plate. The pore structure and collagen in a scaffold may play an important role in fostering the biochemical functions of oval cells. The three-dimensional culture of oval cells could be considered in designing a cell-delivering tool for hepatic disease.     

Characterization of porous collagen/hyaluronic acid scaffold modified by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide cross-linking.

In order to develop a scaffolding material for tissue regeneration, porous matrices containing collagen and hyaluronic acid were fabricated by freeze drying at -20 degrees C, -70 degrees C or -196 degrees C. The fabricated porous membranes were cross-linked using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) in a range of 1-100 mM concentrations for enhancing mechanical stability of the composite matrix. Scanning electron microscope (SEM) views of the matrices demonstrated that the matrices obtained before cross-linking process had interconnected pores with mean diameters of 40, 90 or 230 microm and porosity of 58-66% according to the freezing temperature, and also the porous structures after cross-linking process were retained. The swelling test and IR spectroscopic measurement of different cross-linked membranes were carried out as a measure of the extent of cross-linking. The swelling behavior of cross-linked membranes showed no significant differences as cross-linking degree increased. FT-IR spectra showed the increase of the intensity of the absorbencies at amide bonds (1655, 1546, 1458 cm(-1)) compared to that of CH bond (2930 cm(-1)). In enzymatic degradation test, EDC treated membranes showed significant enhancement of the resistance to collagenase activity in comparison with 0.625% glutaraldehyde treated membranes. In cytotoxicity test using L929 fibroblastic cells, the EDC-cross-linked membranes demonstrated no significant toxicity.     

Hyaluronic acid and hyaluronic acid-binding proteins in brain extracellular matrix

Hyaluronic acid (HA) plays the main structural role in the formation of brain extracellular matrix (ECM). The extracellular space appears empty by electron microscopy because HA is readily dissolved during the preparation of tissues for ultrastructural studies. The HA-binding proteins so far identified in brain ECM are versican, aggrecan and the glial HA-binding protein. Versican is a large fibroblast proteoglycan preferentially expressed in embryonic cartilage at the time of mesenchymal condensation. Glial HA-binding protein (GHAP) is probably a proteolytic product of versican corresponding to its HA-binding amino-terminal domain. It is mainly a white-matter protein, suggesting that the proteinase responsible for its cleavage from versican is normally activated in this location. Versican is found in both white matter and gray matter, where it forms pericellular coats around large neurons. Aggrecan, the aggregating proteoglycan of mature cartilage, co-localizes with versican in this location. In white matter, the localization of GHAP and versican is identical to that of the glial fibrillary acid protein, suggesting that both proteins are produced by astrocytes. An important difference between GHAP and versican is that GHAP but not versican is released from the tissues by hyaluronidase digestion, which suggests that versican is anchored to the cell membranes lining the extracellular space. GHAP was localized at the ultrastructural level in the granule cell layer of rat cerebellum, the only region of gray matter that is positive for GHAP in this species. Rats were perfused with aqueous fixatives containing cetylpyridinium chloride or tannic acid to prevent the solubilization of HA. GHAP is found throughout the extracellular space, the synaptic clefts being a notable exception. GHAP appears late in development, and the same is true for versican, the characteristic perineuronal coats first becoming apparent in the third postnatal week. It is suggested that a marked change occurs in the structure of brain ECM when HA-binding proteins first appear, and that the change is similar to that observed in prechondrogenic mesenchyme, i.e., reduction of the extracellular space and cell aggregation.     

Development of a wound dressing composed of hyaluronic acid and collagen sponge with epidermal growth factor

This study was designed to investigate the effect of a wound dressing composed of hyaluronic acid (HA) and collagen (Col) sponge containing epidermal growth factor (EGF) on various parameters of wound healing in vitro and in vivo. High-molecular-weight (HMW) HA solution, hydrolyzed low-molecular-weight (LMW) HA solution and heat-denatured Col solution were mixed, followed by freeze-drying to obtain a spongy sheet. Cross-linkage between Col molecules was induced by UV irradiation to the spongy sheet (Type-I dressing). In a similar manner, a spongy sheet containing EGF was prepared (Type-II dressing). The efficacy of these products was firstly evaluated in vitro. Fibroblast proliferation was assessed in culture medium in the presence or absence of a piece of each wound dressing. EGF stimulated cell proliferation after UV irradiation and dry sterilization at 110°C for 1 h. In the second experiment, fibroblasts-embedded Col gels were elevated to the air-liquid interface to create a wound surface model, on which wound dressings were placed and cultured for 1 week. Cell proliferation and the production of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were investigated. With Type-II dressings, the amounts of VEGF and HGF released from fibroblasts in the Col gel were significantly increased compared with Type-I dressing. Next, the efficacy of these products was evaluated in vivo using Sprague-Dawley (SD) rats. Wound conditions after 1 and 2 weeks of treatment with the wound dressings were evaluated based on the gross and histological appearances. Type-II dressings promoted a decrease in wound size, re-epithelialization and granulation tissue formation associated with angiogenesis. These findings indicate that the combination of HA, Col and EGF promotes wound healing by stimulating fibroblast function.     

Histological evidence for enhanced anal fistula repair using autologous fibroblasts in a dermal collagen matrix

An anal fistula has a primary track passing from an internal anal or rectal opening to an external opening in the perianal area. Surgery aims to eradicate sepsis whilst preserving faecal continence. Fistulotomy, when all tissue caudal to the primary fistulous track is opened, provides the surest method of cure but may diminish patient continence. An alternative sphincter-preserving procedure is to instill a sealant into the track. An experimental porcine model of fistula-in-ano has been developed in the Surgical Research Department at Northwick Park Institute for Medical Research. This allows histological assessment of fistula tracks after novel, sphincter-preserving surgery and treatments have been applied. Under general anaesthetic, 24 anal fistulae were created and treated, three in each of eight adult Large White/Landrace crossbred pigs. Under the same general anaesthetic, a split skin graft was taken from which to culture fibroblasts for future treatment. All tracks were treated at 4 weeks post-track induction when the tracks were established and very similar in clinical appearance to human tracks. All tracks were prepared for treatment using an instrument designed to remove granulation tissue from the wall of the track. Five control tracks were not infilled but simply had their internal and external openings closed with a Vicryl suture. Nine tracks were treated by infill using an acellular porcine dermal collagen matrix. Ten tracks were treated using a mixture of this matrix and autologous cultured fibroblasts. Histological examination of six tracks was carried out at 2 weeks, nine tracks at 2 months and nine tracks at 3 months. Histological assessment demonstrated persistent fistula tracks in only two fistulae, both of which were control tracks. All treated tracks were closed and cured at all times of examination. When autologous fibroblasts were added to the infill material, cellular integration and vascularization were improved. Using this pre-clinical model, provided fistulous tracks were prepared using a new, in-house developed instrument; treatment with acellular collagen matrix alone healed all tracks. Adding autologous fibroblasts improved the quality of wound healing. A pilot study using this treatment in human fistula patients is in progress. This paper was first presented at the European Histology Forum Annual Conference on 24–26 April 2006.