Synthesis by gamma irradiation of hyaluronic acid-polyvinyl alcohol hydrogel for biomedical applications.

Hyaluronic acid (HA) is one of the most attractive natural polymers employed in biomaterials with biological applications. This polysaccharide is found in different tissues of the body because it is a natural component of the extracellular matrix; furthermore, it has crucial functions in cell growth, migration, and differentiation. Since its biological characteristics, HA has been utilized for the new biomaterial's development for tissue engineering, such as hydrogels. These hydrophilic macromolecular networks have gained significant attention due to their unique properties, making them potential candidates to be applied in biomedical fields. Different mechanisms to obtain hydrogels have been described. However, the research of new non-toxic methods has been growing in recent years. In this study, we prepared a new hydrogel of HA and polyvinyl alcohol by the cost-effective technique of cross-linking by gamma irradiation. The hydrogel was elaborated for the first time and was characterized by several methods such as Fourier Transform Infrared Spectroscopy, Differential Scanning Calorimetry, Thermogravimetric Analysis, and Scanning Electron Microscopy. Likewise, we evaluated the cytotoxicity of the biomaterial and its influence on cell migration in human fibroblasts. Furthermore, we provide preliminary evidence of the wound closure effect in a cellular wound model. The novel hydrogel offers an increase of HA stability with the potential to expand the useful life of HA in its different medical applications.

Enhancement of T2* Weighted MRI Imaging Sensitivity of U87MG Glioblastoma Cells Using γ-Ray Irradiated Low Molecular Weight Hyaluronic Acid-Conjugated Iron Nanoparticles.

INTRODUCTION: It has been reported that low-molecular-weight hyaluronic acid (LMWHA) exhibits a potentially beneficial effect on cancer therapy through targeting of CD44 receptors on tumor cell surfaces. However, its applicability towards tumor detection is still unclear. In this regard, LMWHA-conjugated iron (Fe3O4) nanoparticles (LMWHA-IONPs) were prepared in order to evaluate its application for enhancing the T2* weighted MRI imaging sensitivity for tumor detection. METHODS: LMWHA and Fe3O4 NPs were produced using γ-ray irradiation and chemical co-precipitation methods, respectively. First, LMWHA-conjugated FITC was prepared to confirm the ability of LMWHA to target U87MG cells using fluorescence microscopy. The hydrodynamic size distribution and dispersion of the IONPs and prepared LMWHA-IONPs were analyzed using dynamic light scattering (DLS). In addition, cell viability assays were performed to examine the biocompatibility of LMWHA and LMWHA-IONPs toward U87MG human glioblastoma and NIH3T3 fibroblast cell lines. The ability of LMWHA-IONPs to target tumor cells was confirmed by detecting iron (Fe) ion content using the thiocyanate method. Finally, time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging and in vitro magnetic resonance imaging (MRI) were performed to confirm the contrast enhancement effect of LMWHA-IONPs. RESULTS: Florescence analysis results showed that LMWHA-FITC successfully targeted the surfaces of both tested cell types. The ability of LMWHA to target U87MG cells was higher than for NIH3T3 cells. Cell viability experiments showed that the fabricated LMWHA-IONPs possessed good biocompatibility for both cell lines. After co-culturing test cells with the LMWHA-IONPs, detected Fe ion content in the U87MG cells was much higher than that of the NIH3T3 cells in both thiocyanate assays and TOF-SIMs images. Finally, the addition of LMWHA-IONPs to the U87MG cells resulted in an obvious improvement in T2* weighted MR image contrast compared to control NIH3T3 cells. DISCUSSION: Overall, the present results suggest that LMWHA-IONPs fabricated in this study provide an effective MRI contrast agent for improving the diagnosis of early stage glioblastoma in MRI examinations.

Tumor-targeting hyaluronic acid/fluorescent carborane complex for boron neutron capture therapy.

In cancer therapeutics, boron neutron capture therapy (BNCT) requires a platform for selective and efficient 10B delivery into tumor tissues for a successful treatment. However, the use of carborane, a promising candidate with high boron content and biostability, has significant limitations in the biomedical field due to its poor water-solubility and tumor-selectivity. To overcome these hurdles, we present in this study a fluorescent nano complex, combining fluorescent carborane and sodium hyaluronate for high boron concentration and tumor-selectivity. Tumor cells actively internalized the complex through binding hyaluronan to CD44, overexpressed on the tumor cell surface. Furthermore, the subcellular distribution of this complex could also be detected due to its fluorescent properties. Moreover, after thermal neutron irradiations, the complex produced excellent cytotoxicity, equal to or greater than that of the clinically-used BPA-fructose. Therefore, this novel complex could be potentially more suitable for BNCT than the boron agent.

Extracellular matrix-mimetic composite hydrogels of cross-linked hyaluronan and fibrillar collagen with tunable properties and ultrastructure.

A platform of enzymatically-crosslinked Collagen/Tyramine hyaluronan derivative (Col/HA-Tyr) hydrogels with tunable compositions and gelation conditions was developed to evaluate the impact of the preparation conditions on their physical, chemical and biological properties. At low HA-Tyr content, hydrogels exhibited a fibrillar structure, with lower mechanical properties compared to pure Col hydrogels. At high HA-Tyr and Horse Radish Peroxydase (HRP) content, a microfibrillar network was formed beside the banded Col fibrils and a synergistic effect of the hybrid structure on mechanical properties was observed. These hydrogels were highly resistant against enzymatic degradation while keeping a high degree of hydration. Unlike HA-Tyr hydrogels, encapsulation of human dermal fibroblasts within Col/HA-Tyr hydrogels allowed for high cell viability. These results showed that high HA-Tyr and HRP concentrations are required to positively impact the physical properties of hydrogels while preserving collagen fibrils. Those Col/HA-Tyr hydrogels appear promising for novel tissue engineering applications following a biomimetic approach.

Biofunctions of antimicrobial peptide-conjugated alginate/hyaluronic acid/collagen wound dressings promote wound healing of a mixed-bacteria-infected wound.

The increase in severe infections caused by antibiotic drug resistance and the decrease in the number of new antibacterial drugs approved for use in the last few decades are driving the need for the development of new antimicrobial strategies. Antimicrobial peptides (AMPs) are a potential new class of antimicrobial drugs that are expected to solve the problem of global antibiotic drug resistance. Herein, the AMP Tet213 was immobilised onto the substrates of alginate (ALG), hyaluronic acid (HA), and collagen (COL) to form the ALG/HA/COL-AMP wound dressing. This wound dressing exhibited a high degree of swelling and the appropriate porosity, mechanical properties, and biodegradability. The Tet213-immobilised ALG/HA/COL dressings exhibited antimicrobial activity against three pathogenic bacterial strains (Gram-negative E. coli and Gram-positive MRSA and S. aureus) and facilitated the proliferation of NIH 3T3 fibroblast cells. In addition, the ALG/HA/COL-AMP antimicrobial dressings promoted wound healing, re-epithelialisation, collagen deposition, and angiogenesis. Moreover, the wound-healing effects of ALG/HA/COL-AMP surpassed the gauze and ALG/HA/COL compared to commercially available silver-based dressings (Aguacel Ag). These results suggest that the Tet213-conjugated ALG/HA/COL wound dressing, with its multiple biological activities, is a promising wound-dressing material.

Tumor Elastography and Its Association with Collagen and the Tumor Microenvironment.

Purpose: The tumor microenvironment presents with altered extracellular matrix (ECM) and stroma composition, which may affect treatment efficacy and contribute to tissue stiffness. Ultrasound (US) elastography can visualize and quantify tissue stiffness noninvasively. However, the contributions of ECM and stromal components to stiffness are poorly understood. We therefore set out to quantify ECM and stroma density and their relation to tumor stiffness.Experimental Design: A modified clinical ultrasound system was used to measure tumor stiffness and perfusion during tumor growth in preclinical tumor models. In vivo measurements were compared with collagen mass spectroscopy and automatic analysis of matrix and stromal markers derived from immunofluorescence images.Results: US elastography estimates of tumor stiffness were positively correlated with tumor volume in collagen and myofibroblast-rich tumors, while no correlations were found for tumors with low collagen and myofibroblast content. US elastography measurements were strongly correlated with ex vivo mechanical testing and mass spectroscopy-based measurements of total collagen and immature collagen crosslinks. Registration of ultrasound and confocal microscopy data showed strong correlations between blood vessel density and T-cell density in syngeneic tumors, while no correlations were found for genetic tumor models. In contrast to collagen density, which was positively correlated with stiffness, no significant correlations were observed for hyaluronic acid density. Finally, localized delivery of collagenase led to a significant reduction in tumor stiffness without changes in perfusion 24 hours after treatment.Conclusions: US elastography can be used as a potential biomarker to assess changes in the tumor microenvironment, particularly changes affecting the ECM. Clin Cancer Res; 24(18); 4455-67. ©2018 AACR.

Hyaluronic acid decreases the mechanical stability, but increases the lytic resistance of fibrin matrices.

Hyaluronic acid (HA) is a large, non-sulfated glucosaminoglycan abundantly present at sites where fibrin is also formed (during wound healing, in arterial restenotic lesions and eroded atherosclerotic plaques). The aim of the present study was to characterize the structure of composite fibrin-HA clots with scanning electron microscopy (SEM), pressure-driven permeation and small-angle X-ray scattering (SAXS) and their viscoelastic properties with an oscillation rheometer. In addition the efficiency of fibrinolysis in these clots was investigated by kinetic turbidimetric and chromogenic assays for dissolution of fibrin and plasminogen activation by tissue-type plasminogen activator (tPA). Fibrin formed in the presence of native (1500kDa) HA and its 500kDa fragments had thicker fibers and larger pores according to the SEM and clot permeation data, whereas the 25kDa HA fragments had only minor effects. SAXS evidenced a mild disarrangement of protofibrils. These structural alterations suggest that HA modifies the pattern of fibrin polymerization favouring lateral association of protofibrils over formation of branching points. Rheometer data showed softer fibrin structures formed with 1500kDa and 500kDa HA and these clots presented with lower dynamic viscosity values and lower critical stress values at gel/fluid transition. tPA-catalysed plasminogen activation was markedly inhibited by HA, both in free solution and on the surface of fibrin clots, in the presence and in the absence of 6-aminohexanoate suggesting a kringle-independent mechanism. HA of 1500 and 500kDa size prolonged clot lysis with both plasmin and tPA and this inhibition was kringle-mediated, because it was abolished by 6-aminohexanoate and was not observed with des-(kringle1-4)-plasmin. Our data suggest that HA size-dependently modifies the pattern of fibrin polymerization with consequent inhibition of fibrinolysis. At sites of tissue injury and inflammation, HA could stabilize fibrin through modification of its structure and lysibility.

Application of hyaluronic acid/sodium alginate-based microparticles to prevent tissue adhesion in a rabbit model.

PURPOSE: Postsurgical adhesion formation is a concern in every field of surgery. We evaluated the efficacy of hyaluronic acid/sodium alginate-based microparticle anti-adhesive agents (MP) for the prevention of postsurgical adhesion formation in a standardized rabbit model. METHOD: To evaluate the anti-adhesion effect, a uterus-abdominal wall abrasion model was created in rabbits. On the surface of the injured uterus, an anti-adhesive agent, Interceed(®) or MP, was applied (positive control and study groups, respectively; n = 10 each). In another group of 10 animals, neither agent was applied (negative control group). The adhesion levels were graded 3 weeks after surgery. Acute and chronic toxicity was also evaluated. RESULTS: The grade of adhesion was significantly lower in the MP group than in the negative control and positive control groups. No evidence of acute or chronic toxicity induced by this material was found in blood and tissue analysis. CONCLUSION: MP shows potential as an effective novel type of resorbable biomaterial to reduce postoperative adhesion. The easy placement and handling of this material make the MP powder attractive as a tissue adhesion barrier.

Ultrastructural analysis of 3 hyaluronic acid soft-tissue fillers using scanning electron microscopy.

BACKGROUND: Although hyaluronic acid (HA) specifications such as molecular weight and particle size are fairly well characterized, little information about HA ultrastructural and morphologic characteristics has been reported in clinical literature. OBJECTIVE: To examine uniformity of HA structure, the effects of extrusion, and lidocaine dilution of 3 commercially available HA soft-tissue fillers. MATERIALS AND METHODS: Using scanning electron microscopy and energy-dispersive x-ray analysis, investigators examined the soft-tissue fillers at various magnifications for ultrastructural detail and elemental distributions. RESULTS: All HAs contained oxygen, carbon, and sodium, but with uneven distributions. Irregular particulate matter was present in RES but BEL and JUV were largely particle free. Spacing was more uniform in BEL than JUV and JUV was more uniform than RES. Lidocaine had no apparent effect on morphology; extrusion through a 30-G needle had no effect on ultrastructure. CONCLUSION: Descriptions of the ultrastructural compositions and nature of BEL, JUV, and RES are helpful for matching the areas to be treated with the HA soft-tissue filler architecture. Lidocaine and extrusion through a 30-G needle exerted no influence on HA structure. Belotero Balance shows consistency throughout the syringe and across manufactured lots.

Influence of water content and drying on the physical structure of native hyaluronan.

Hydration properties of semi-diluted hyaluronan were studied by means of time domain nuclear magnetic resonance. Based on the transverse proton relaxation times T2, the plasticization of hyaluronan which was precipitated by isopropylalcohol and dried in the oven have been determined at water content 0.4 g of water per g of hyaluronan. Above this water content, the relaxation times increased and levelled off around 0.8 g of water per g of hyaluronan which agrees well with values determined earlier by differential scanning calorimetry and dielectric relaxometry. The freeze dried and oven dried samples showed differences in their physical structure such as glass transition, plasticization concentration and sample topography which influenced their kinetics and mechanisms of hydration. Results confirmed earlier hypothesis that some native biopolymer structures can be easily modified by manipulation of preparation conditions, e.g. drying, giving fractions with specific physicochemical properties without necessity of their chemical modification.

Photo-crosslinked hyaluronic acid nanoparticles with improved stability for in vivo tumor-targeted drug delivery.

One of the major hurdles of the nanoparticles as drug carriers is the unintended burst release of loaded drugs during blood circulation. To surmount this issue, we developed photo-crosslinked hyaluronic acid nanoparticles (c-HANPs) with improved stability for tumor-targeted drug delivery. They were readily prepared via UV-triggered chemical crosslinking with the acrylate groups in the polymer backbone. The size of c-HANPs was not much different from that of uncrosslinked HANPs. However, c-HANPs exhibited significantly high stability in a physiological buffer and released the loaded drug, paclitaxel (PTX), in a sustained manner. It is noteworthy that the drug release rate from c-HANPs remarkably increased in the presence of hyaluronidase, an enzyme abundant at the intracellular compartments of the tumor cells. It was found from in vitro cellular uptake tests that c-HANPs were rapidly taken up by the tumor cells via the receptor (CD44)-mediated endocytosis, which was not inhibited by photo-crosslinking. In non-invasive animal imaging results, they showed higher tumor-targeting ability than uncrosslinked HANPs because high stability of c-HANPs enabled their long circulation in the body. Owing to the sustained release of the drug and enhanced tumor-targeting ability, c-HANPs showed higher therapeutic efficacy compared to free PTX and uncrosslinked HANPs. These data implied the promising potential of c-HANP as tumor-targeting drug carriers and demonstrated the remarkable effect of the improved stability upon the biodistribution and therapeutic efficacy of drug-loaded nanoparticles.

Nanofibers in a hyaluronan-based pericellular matrix.

The extracellular matrix of the brain is a highly organized hyaluronan-based supramolecular assembly that is involved in neuronal pathfinding, cell migration, synaptogenesis and neuronal plasticity. Here, we analyze the structure of the hyaluronan-rich pericellular matrix of an oligodendroglial precursor cell line using helium ion beam scanning microscopy at a subnanometer resolution. We find that thin nanofibers are the ultimate building elements of this oligodendroglial pericellular matrix. These structures may participate in the regulation of oligodendroglial maturation and motility.

Adsorption of albumin and sodium hyaluronate on UHMWPE: a QCM-D and AFM study.

The biotribological properties of artificial joints, in particular the efficiency of the lubrication, strongly determine their lifetime. The most commonly used artificial joints combine a metallic or ceramic part articulating against a ultra high molecular weight polyethylene (UHMWPE) counterface, and are lubricated by the periprosthetic fluid. This fluid contains several macromolecules, namely albumin and sodium hyaluronate (NaHA), that are known to be involved in the lubrication process. There are several studies in the literature concerning the interaction of the referred macromolecules with ceramic or metallic prosthetic materials. However, to our knowledge, information about their binding to the polymeric surface is practically inexistent. The objective of this work is to contribute to clarify the role played by albumin and NaHA on the biolubrication process, through the investigation of their interaction with the UHMWPE surface. The study involves adsorption measurements using a quartz crystal microbalance with dissipation (QCM-D), the characterization of the adsorbed films by atomic force microscopy (AFM) and wettability determinations. Albumin was found to adsorb strongly and extensively to the polymer, while NaHA led to a very low adsorption. In both cases rigid films were obtained, but with different morphology and porosity. The high binding affinity of the protein to the polymer was demonstrated both by the results of the fittings to Langmuir and Freundlich models and by the values of the adhesion forces determined by AFM. In the simultaneous adsorption of albumin and NaHA, protein adsorption is predominant and determines the surface properties.

Single-molecule imaging of hyaluronan in human synovial fluid.

Human synovial fluid contains a high concentration of hyaluronan, a high molecular weight glycosaminoglycan that provides viscoelasticity and contributes to joint lubrication. In osteoarthritis synovial fluid, the concentration and molecular weight of hyaluronan decrease, thus impairing shock absorption and lubrication. Consistently, substitution of hyaluronan (viscosupplementation) is a widely used treatment for osteoarthritis. So far, the organization and dynamics of hyaluronan in native human synovial fluid and its action mechanism in viscosupplementation are poorly characterized at the molecular level. Here, we introduce highly sensitive single molecule microscopy to analyze the conformation and interactions of fluorescently labeled hyaluronan molecules in native human synovial fluid. Our findings are consistent with a random coil conformation of hyaluronan in human synovial fluid, and point to specific interactions of hyaluronan molecules with the synovial fluid matrix. Furthermore, single molecule microscopy is capable of detecting the breakdown of the synovial fluid matrix in osteoarthritis. Thus, single molecule microscopy is a useful new method to probe the structure of human synovial fluid and its changes in disease states like osteoarthritis.

Ultrastructural and immunohistochemical analysis of fibrous long-spacing collagen fibrils in malignant mesothelioma.

Three cases of biphasic mesothelioma and 2 cases of sarcomatoid mesothelioma were investigated using light and electron microscopy. In 2 of the 3 cases of biphasic mesotheliomas, fibrous long-spacing (FLS) collagen fibrils were discovered with a symmetrical cross-striation of 130 nm in periodicity. However, no connection between the FLS fibrils and usual collagen fibrils were observed. Periodic acid silver methenamine stain revealed unstained bands with periods of 130 nm in FLS fibrils, whereas the usual collagen fibrils showed continuous positive staining. All 3 cases of biphasic mesotheliomas showed deposits of hyaluronic acid, whereas both cases of sarcomatoid mesotheliomas showed little hyaluronic acid. As a high concentration of hyaluronic acid induces the formation of FLS collagen fibrils in vitro, the authors propose that FLS fibrils from mesothelioma may be special structures that occur as the tropocollagens are assembled into new collagen fibrils in the presence of hyaluronic acid.

Influences of construct properties on the proliferation and matrix synthesis of dedifferentiated chondrocytes cultured in alginate gel.

To investigate whether the chondrocytes-alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3 x 10(6) cells/ml) or high density (HD: 10 x 10(6) cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell-cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes' redifferentiation and should be considered for creating of an appropriate engineered articular cartilage.

In vivo real-time bioimaging of hyaluronic acid derivatives using quantum dots.

The effect of chemical modification of hyaluronic acid (HA) on its distribution throughout the body was successfully visualized in nude mice through real-time bioimaging using quantum dots (QDots). Adipic acid dihydrazide modified HA (HA-ADH) was synthesized and conjugated with QDots having carboxyl terminal ligands activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide. The formation of HA-QDot conjugates could be confirmed by gel permeation chromatography, fluorometry, transmission electron microscopy, and zeta-size analysis. According to the real-time bioimaging of HA-QDot conjugates after subcutaneous injection to nude mice, the fluorescence of HA-QDot conjugates with a near infrared wavelength of 800 nm could be detected up to 2 months, whereas that with an emission wavelength of 655 nm disappeared almost completely within 5 days. The results can be ascribed to the fact that near-infrared light has a high penetration depth of about 5-6 cm in the body compared to that of about 7-10 mm for visible light. Thereby, using QDots with a near-infrared emission wavelength of 800 nm, the distribution of HA-QDot conjugates throughout the body was bioimaged in real-time after their tail-vein injection into nude mice. HA-QDot conjugates with 35 mol% ADH content maintaining enough binding sites for HA receptors were mainly accumulated in the liver, while those with 68 mol% ADH content losing much of HA characteristics were evenly distributed to the tissues in the body. The results are well matched with the fact that HA receptors are abundantly present in the liver with a high specificity to HA molecules.

Induction of hyaluronan cables and monocyte adherence in epidermal keratinocytes.

Hyaluronan attached to cell surface can form at least two very different structures; a pericellular coat close to plasma membrane and hyaluronan chains coalesced into "cables" that can span several cell lengths. The hyaluronan in cables, induced by many inflammatory agents, can bind leukocytes, whereas that in the pericellular coat does not contribute to leukocyte binding. Therefore, this structural change seems to have a major role in inflammation. In the present study we checked whether cells of squamous epithelium, like epidermal keratinocytes, can form hyaluronan cables and bind leukocytes. In addition, we checked whether hyaluronan synthesis is affected during the induction of cables. Control keratinocytes expressed pericellular hyaluronan as small patches on plasma membrane. But when treated with inflammatory agents or stressful conditions (tunicamycin, interleukin-1beta, tumor necrosis factor-alpha, and high glucose concentration), hyaluronan organization changed into cable-like structures that avidly bound monocytes. Simultaneously, the total amount of secreted hyaluronan was slightly decreased, and the expression levels of hyaluronan synthases (Has1-3) and CD44 were not significantly changed. The results show that epidermal keratinocytes can form cables and bind leukocytes under inflammatory provocation and that these effects are not dependent on stimulation of hyaluronan secretion.

Mechanically strong double network photocrosslinked hydrogels from N,N-dimethylacrylamide and glycidyl methacrylated hyaluronan.

Hyaluronan (HA) is a natural polysaccharide abundant in biological tissues and it can be modified to prepare biomaterials. In this work, HA modified with glycidyl methacrylate was photocrosslinked to form the first network (PHA), and then a series of highly porous PHA/N,N-dimethylacrylamide (DAAm) hydrogels (PHA/DAAm) with high mechanical strength were obtained by incorporating a second network of photocrosslinked DAAm into PHA network. Due to the synergistic effect produced by double network (DN) structure, despite containing 90% of water, the resulting PHA/DAAm hydrogel showed a compressive modulus and a fracture stress over 0.5 MPa and 5.2 MPa, respectively. Compared to the photocrosslinked hyaluronan single network hydrogel, which is generally very brittle and fractures easily, the PHA/DAAm hydrogels are ductile. Mouse dermal fibroblast was used as a model cell line to validate in vitro non-cytotoxicity of the PHA/DAAm hydrogels. Cells deposited extracellular matrix on the surface of these hydrogels and this was confirmed by positive staining of Type I collagen by Sirius Red. The PHA/DAAm hydrogels were also resistant to biodegradation and largely retained their excellent mechanical properties even after 2 months of co-culturing with fibroblasts.

Regeneration of dentin-pulp complex with cementum and periodontal ligament formation using dental bud cells in gelatin-chondroitin-hyaluronan tri-copolymer scaffold in swine.

The purpose of this study is to use a tissue engineering approach for tooth regeneration. The swine dental bud cells (DBCs) were isolated from the developing mandibular teeth, expanded in vitro, and cultured onto cylinder scaffold gelatin-chrondroitin-hyaluronan-tri-copolymer (GCHT). After culturing in vitro, the DBCs/GCHT scaffold was autografted back into the original alveolar socket. Hematoxylin and eosin (H&E) staining combined with immunohistochemical staining were applied for identification of regenerated tooth structure. After 36-week post-transplantation, tooth-like structures, including well-organized dentin-pulp complex, cementum, and periodontal ligament, were evident in situ in two of six experimental animals. The size of the tooth structure (1 x 0.5 x 0.5 cm(3) and 0.5 x 0.5 x 0.5 cm(3) size) appeared to be dictated by the size of the GCHT scaffold (1 x 1 x 1.5 cm(3)). The third swine was demonstrated with irregular dentin-bony like calcified tissue about 1 cm in diameter without organized tooth or periodontal ligament formation. The other three swine in the experimental group showed normal bone formation and no tooth regeneration in the transplantation sites. The successful rate of tooth regeneration from DBCs/GCHT scaffolds' was about 33.3%. In the control group, three swine's molar teeth buds were removed without DBCs/GCHT implantation, the other three swine received GCHT scaffold implants without DBCs. After evaluation, no regenerated tooth was found in the transplantation site of the control group. The current results using DBSs/GCHT scaffold autotransplantation suggest a technical breakthrough for tooth regeneration.