Biomechanical evaluation of a novel tri-blade titanium implantable vertebral augmentation device.

BACKGROUND CONTEXT: Titanium implantable vertebral augmentation device (TIVAD) are regarded as having potential in the treatment of vertebral compression fractures (VCFs). However, improper design in current TIVADs results in the inability to effectively restore VCF height and maintain stability. There is still an unmet clinical need for improvement. PURPOSE: The authors tested a newly developed a TIVAD (Tri-blade fixed system) that can provide enough endplate collapse support to restore the vertebral body height in a safe retraction mechanism for VCFs using minimally invasive surgery (MIS). STUDY DESIGN: The performed biomechanical tests included blade expansion force, lifetime of cement embedded and vertebral height restoration efficiency of porcine osteoporosis VCFs for its feasibility. METHODS: A cylinder with 3 surface cuts that form blades that can be expanded into a conical space was designed (Tri-blade fixed system). The 3 blades can be expanded outward with angles between blades as 105°/ 105°/150° for lower left/lower right/upper arms, respectively that reach 15mm in height and 14.8 mm in width. A frame was specifically designed to measure the contact force using force sensing resistors during blade expansion. The Tri-blade fixed system was embedded into a cement block to perform fatigue testing under 2000N pressure (5*106 cycles) for understanding the device lifetime limitation. The Tri-blade system was then inserted into porcine osteoporosis VCFs to examine the vertebral height restoration efficiency. RESULTS: The average maximum contact force for the top, bottom left and right blades were 299.0N, 283.5N and 279.3N, respectively with uniformly outward expansion forces. The fatigue test found that there were no obvious cracks or damage to the cement block. The porcine osteoporosis vertebral body at the anterior, middle, and posterior heights can be restored to 21.9%, 12.6% and 6.4%, respectively. CONCLUSIONS: This study developed a novel TIVAD with conical shape that can provide a more stable structure with sufficient/uniform expansion force, passing the fatigue test with bone cement and high effective in vertebral height restoration tests for porcine osteoporosis VCFs. CLINICAL SIGNIFICANCE: The new 3D Tri-blade TIVAD may offer a new treatment option for VCFs.

The efficacy of vancomycin-loaded biphasic calcium phosphate bone substitute in the promotion of new bone growth and the prevention of postoperative infection.

Background: Due to the increasing need for suitable alternatives to bone grafts, artificial bones made of biphasic calcium phosphate (BCP) are currently being extensively researched. These porous bone substitutes have also demonstrated considerable incorporation with the host bone, and new bone is able to grow within the porous structure. They therefore offer a potential therapeutic approach for bone defects. Methods: Vancomycin-loaded Bicera™, a BCP bone substitute, was investigated in order to prevent implant-associated osteomyelitis and postoperative infection after orthopedic surgery. The loading capacity of Bicera™ was measured to understand its potential antibiotic adsorption volume. An antibiotic susceptibility test was also carried out to analyze the effect of Bicera™ loaded with different concentrations of vancomycin on the growth inhibition of methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin-loaded Bicera™ was implanted into rabbits with bone defects, and general gross, radiographic, and histological evaluation was undertaken at 4, 12, and 24 weeks after implantation. Results: The maximum loading capacity of vancomycin-loaded Bicera™ was 0.9 ml of liquid regardless of the vancomycin concentration. Antibiotic susceptibility tests showed that vancomycin-loaded Bicera™ inhibited the growth of MRSA for 6 weeks. In addition, animal studies revealed that new bone grew into the vancomycin-loaded Bicera™. The percentage of new bone formation from 4 to 24 weeks after implantation increased from 17% to 36%. Conclusion: Vancomycin-loaded Bicera™ could effectively inhibit the growth of MRSA in vitro. It was found to incorporate into the host bone well, and new bone was able to grow within the bone substitute. The results of this study indicate that vancomycin-loaded Bicera™ is a potential bone substitute that can prevent implant-associated osteomyelitis and postoperative infection.

A novel surgical technique in transforaminal lumbar interbody fusion by the bone graft delivery device: evaluation of therapeutic effect in patients with minimally invasive spine surgery.

BACKGROUND: Transforaminal Lumbar Interbody Fusion (TLIF) is commonly associated with higher complications and longer operative time. This study aims to evaluate the effectiveness, safety, and usability of a novel minimally invasive surgery (MIS) bone graft delivery device. METHODS: 73 consecutive patients with lumbar spondylosis, degenerative disc disease, spondylolisthesis, scoliosis or trauma were enrolled in this randomized controlled trial. Group 1 comprised 39 patients treated with the novel MIS bone graft delivery device. Group 2 consisted of 34 patients treated with the conventional system. The primary objective of the study was the assessment of the amount of bone graft delivery using the device. The secondary objectives were the effect of the device on operative time, pain relief, disability improvement, and bone fusion grade. RESULTS: Bone delivery amount was significantly higher in the MIS device group (6.7 ± 2.9 mL) compared to the conventional group (2.3 ± 0.5 mL), p < 0.001. Regarding the operation time, the MIS device group was associated significantly lower duration than the conventional group (p < 0.001). After a 3-month follow-up, 39.5% of the patients in the MIS device group and 3.5% of the patients in the conventional group were observed to achieve grade I fusion (complete fusion). There was a significant difference in fusion success rates (p < 0.01). CONCLUSION: The novel MIS bone graft delivery device was associated with successful bone delivery. Our MIS device provides promising modality with less operative time and higher bone fusion rates than conventional modalities. Trial Registration This trial was retrospectively registered on ClinicalTrials.gov (Registration date: 11/19/2021; Registration number: NCT05190055).

Clinical Efficacy of Polycaprolactone ß-Calcium Triphosphate Composite for Osteoconduction in Rabbit Bone Defect Model.

The combination of ß-tricalcium phosphate (ß-TCP) with polycaprolactone (PCL) has been considered a promising strategy for designing scaffolds for bone grafting. This study incorporated PCL with commercially available ß-TCP (OsteoceraTM) to fabricate an injectable bone substitute and evaluate the effect of PCL on compressive strength and setting time of the hydraulic cement. The mechanical testing was compliant with the ASTM D695 and ASTM C191-13 standards. Results showed that PCL-TCP composite presented a well-defined architecture with uniform pore distribution and a significant increase in compressive strength compared with ß-TCP alone. Eighteen rabbits, each with two surgically created bone defects, were treated using the PCL-TCP composites. The composite materials were resorbed and replaced by newly formed bone tissue. Both PCL-TCP and ß-TCP demonstrated equivalent clinical effects on osteoconduction property in terms of the percentage of newly formed bone area measured by histomorphometric analysis. PCL-TCP was proven to be as effective as the commercially available ß-TCP scaffold (OsteoceraTM).

Bone formation following sinus grafting with an alloplastic biphasic calcium phosphate in Lanyu Taiwanese mini-pigs.

BACKGROUND: To evaluate the new bone formation after grafting with a synthetic biphasic calcium phosphate in sinuses with minimal bone height, the alloplastic and xenograft materials were compared after grafting into Lanyu Taiwanese mini-pig sinuses via split-mouth design. METHODS: In six mini-pigs, synthetic hydroxyapatite/tricalcium phosphate (HA/TCP) particles were inserted into one of the sinus cavities using the extra-oral approach, where deproteinized bovine bone mineral (DBBM) particles were placed contralaterally. Fluorescent bony labels of Alizarin and Calcein green were delivered at weeks 4 and 8, respectively. Animals were sacrificed at week 12 and the augmented tissues were evaluated by cone-beam computed tomography, microcomputed tomography, and histology. RESULTS: By radiographic examination, the mean thicknesses of sinus cortexes for DBBM and HA/TCP groups were similar (0.35 versus 0.38 cm) and the mean volumes augmented were also indifferent (1.29 versus 1.64 cm3 ). The distributions of bones, residual particles, and non-mineralized tissues in augmented masses between groups were undistinguishable. Under microscopy, however, macroporosities of osteons were filled with HA/TCP residual particles, whereas the newly formed bones lay on top of DBBM particle surfaces. Although the mineral deposition rates between groups were indifferent, the mean labeled surface in the HA/TCP group was significantly greater than those in the DBBM group at week 4 (35.16% versus 14.00% for HA/TCP and DBBM, respectively) but less than that at week 8 (19.33% versus 39.16%, respectively). CONCLUSION: Sinus augmentation with synthetic HA/TCP and DBBM exhibited similar effectiveness in new bone formation.

Augmented biosynthesis of cadmium sulfide nanoparticles by genetically engineered Escherichia coli.

Microorganisms can complex and sequester heavy metals, rendering them promising living factories for nanoparticle production. Glutathione (GSH) is pivotal in cadmium sulfide (CdS) nanoparticle formation in yeasts and its synthesis necessitates two enzymes: gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS). Hereby, we constructed two recombinant E. coli ABLE C strains to over-express either gamma-GCS or GS and found that gamma-GCS over-expression resulted in inclusion body formation and impaired cell physiology, whereas GS over-expression yielded abundant soluble proteins and barely impeded cell growth. Upon exposure of the recombinant cells to cadmium chloride and sodium sulfide, GS over-expression augmented GSH synthesis and ameliorated CdS nanoparticles formation. The resultant CdS nanoparticles resembled those from the wild-type cells in size (2-5 nm) and wurtzite structures, yet differed in dispersibility and elemental composition. The maximum particle yield attained in the recombinant E. coli was approximately 2.5 times that attained in the wild-type cells and considerably exceeded that achieved in yeasts. These data implicated the potential of genetic engineering approach to enhancing CdS nanoparticle biosynthesis in bacteria. Additionally, E. coli-based biosynthesis offers a more energy-efficient and eco-friendly method as opposed to chemical processes requiring high temperature and toxic solvents.

Acellular biological tissues containing inherent glycosaminoglycans for loading basic fibroblast growth factor promote angiogenesis and tissue regeneration.

It was found in our previous study that acellular tissues derived from bovine pericardia consist primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans (GAGs). It is speculated that the inherent GAGs in acellular tissues may serve as a reservoir for loading basic fibroblast growth factor (bFGF) and promote angiogenesis and tissue regeneration. This study was therefore designed to investigate effects of the content of GAGs in acellular bovine pericardia on the binding of bFGF and its release profile in vitro while its stimulation in angiogenesis and tissue regeneration in vivo were evaluated subcutaneously in a rat model. To control the content of GAGs, acellular tissues were treated additionally with hyaluronidase for 1 (Hase-D1), 3 (Hase-D3), or 5 days (Hase-D5). The in vitro results indicated that a higher content of GAGs in the acellular tissue resulted in an increase in bFGF binding and in a more gradual and sustained release of the growth factor. The in vivo results obtained at 1 week postoperatively showed that the density and the depth of neo-vessels infiltrated into the acellular tissue loaded with bFGF (acellular/bFGF) were significantly greater than the other test samples. At 1 month postoperatively, vascularized neo-connective tissues were found to fill the pores within each test sample, particularly for the acellular/bFGF tissue. These results suggested that the sustained release of bFGF from the acellular/ bFGF tissue continued to be effective in enhancing angiogenesis and generation of new tissues. In conclusion, the inherent GAGs present in acellular tissues may be used for binding and sustained release of bFGF to enhance angiogenesis and tissue regeneration.

Tissue regeneration observed in a porous acellular bovine pericardium used to repair a myocardial defect in the right ventricle of a rat model.

OBJECTIVE: Nonliving synthetic materials have been widely used to repair myocardial defects; however, material-related failures do occur. To overcome these problems, an acellular bovine pericardium with a porous structure fixed with genipin (the AGP patch) was developed. METHODS: The AGP patch was used to repair a surgically created myocardial defect in the right ventricle of a rat model. A commercially available expanded polytetrafluoroethylene (e-PTFE) patch was used as a control. At retrieval, a computerized mapping system was used to acquire local epicardial electrograms of each implanted sample, and the appearance of each retrieved sample was grossly examined. The retrieved samples were then processed for histologic examination. RESULTS: The amplitude of local electrograms on the AGP patch increased significantly with increasing implantation duration, whereas only low-amplitude electrograms were observed on the e-PTFE patch throughout the entire course of the study. No aneurysmal dilation of the implanted patches was seen for either studied group. Additionally, no tissue adhesion was observed on the outer (epicardial) surface of the AGP patch, whereas a moderate tissue adhesion was observed on the e-PTFE patch. On the inner (endocardial) surface, intimal thickening was observed for both studied groups; however, no thrombus formation was found. Intact layers of endothelial and mesothelial cells were identified on the inner and outer surfaces of the AGP patch, respectively. At 4 weeks postoperatively, smooth muscle cells, together with neomuscle fibers (with a few neocollagen fibrils), neoglycosaminoglycans, and neocapillaries, were observed to fill the pores in the AGP patch, an indication of tissue regeneration. These observations were more pronounced at 12 weeks postoperatively. In contrast, no apparent tissue regeneration was observed in the e-PTFE patch. CONCLUSION: The present study indicated that the AGP patch holds promise to become a suitable patch for surgical repair of myocardial defects.

Cell-free xenogenic vascular grafts fixed with glutaraldehyde or genipin: in vitro and in vivo studies.

Chronic rejection of arterial xenografts results in aneurysmal dilation, due to immune mediated processes. To minimize the immunologic degradation of the graft, a cell-extraction process employing sodium dodecyl sulfate (SDS) was used in the study to remove the cellular components in bovine carotid arteries. To further reduce their immunogenicity, the acellular arteries were fixed with glutaraldehyde (A-GA) or genipin (A-GP). The in vitro properties of all test samples were analyzed. Additionally, the in vivo performance of the heparinized A-GA and A-GP grafts (H-A-GA and H-A-GP) was evaluated in a canine model. It was found that the SDS treatment effectively removed cells from the arterial wall, but the main structures of the extracellular matrix were preserved with a portion of the water-soluble glycosaminoglycans removed. After cell extraction, the elastic lamellae in the media became straightened, and thus made the tissue less extensile. The heparinized tissues significantly reduced platelet adhesion. At retrieval, all implanted grafts were patent and not dilated. Chronic inflammatory response surrounding the implants was observed. However, fixation of acellular tissues by glutaraldehyde or genipin inhibited immune cell penetration into the media and limited tissue degradation, and therefore prevented the arterial wall from dilation. Nevertheless, the H-A-GP graft was superior to the H-A-GA graft in completeness of endothelialization on its luminal surface, and thus precluded thrombus formation.

Loading of a novel angiogenic agent, ginsenoside Rg1 in an acellular biological tissue for tissue regeneration.

In this study, the effects of ginsenoside Rg1, a natural compound isolated from Panax ginseng, on human umbilical vein endothelial cell (HUVEC) behavior in vitro, and on angiogenesis and tissue regeneration in genipin-fixed acellular tissue (extracellular matrix, ECM) in vivo, were investigated. Basic fibroblast growth factor (bFGF) was used as a control. The in vitro results indicated that in the presence of bFGF or Rg1, HUVEC proliferation was significantly increased. Both bFGF and Rg1 promoted HUVEC migration in a Transwell plate assay. In addition, bFGF or Rg1 significantly increased the formation of capillary-like network by HUVECs on Matrigel. Thus, both bFGF and Rg1 enhanced multiple components of angiogenic activity in vitro. The in vivo results obtained 1 week postoperatively showed that the extent of angiogenesis in ECMs was significantly enhanced by bFGF or Rg1. At 1 month postoperatively, vascularized neoconnective tissues were found to fill the pores within ECMs loaded with bFGF or Rg1. There was a significant increase in neocapillary density from 1 week to 1 month for ECMs loaded with Rg1, whereas that observed in ECMs loaded with bFGF stayed approximately the same because of the limitations of protein stability. These results suggested that Rg1 may be a new class of angiogenic agent and may be loaded in ECMs to accelerate tissue regeneration.

Peritoneal regeneration induced by an acellular bovine pericardial patch in the repair of abdominal wall defects.

BACKGROUND: This study was to evaluate the feasibility of using an acellular bovine pericardium fixed with genipin (AGP) to repair an abdominal wall defect created in a rat model. MATERIALS AND METHODS: The glutaraldehyde-fixed acellular pericardium (AGA), the genipin-fixed cellular pericardium (GP), and a commercially available polypropylene mesh were used as controls. RESULTS: Gross examination at 3-month post-operatively revealed that dense adhesions to the visceral organs were observed for the polypropylene mesh and the AGA patch, while a filmy to dense adhesion was seen for the GP patch. In contrast, no adhesion to the visceral organs was observed for the AGP patch. Histologically, inflammatory cells were found mainly surrounding the GP patch. In contrast, host cells (inflammatory cells, fibroblasts, and neo-capillaries) were able to infiltrate into the AGA and AGP patches. Unlike the AGA patch, the AGP patch retrieved at 1-month post-operatively became well integrated with the host tissue near the suture line. Additionally, there were some mesothelial cells, identified by the van Gieson stain, observed on the AGP patch. At 3-month post-operatively, a neo-peritoneum was observed on the AGP patch. The neo-peritoneum consisted of organized vascularized connective tissues covered by an intact layer of mesothelial cells. The calcium contents of the polypropylene mesh and the AGA patch increased significantly at 3-month post-operatively, while those of the GP and AGP patches stayed minimal throughout the entire course of the study. CONCLUSIONS: The results obtained in the study revealed that the AGP patch effectively repaired abdominal wall defects in rats and successfully prevented the formation of post-surgical abdominal adhesions.

A natural compound (ginsenoside Re) isolated from Panax ginseng as a novel angiogenic agent for tissue regeneration.

PURPOSE: The primary challenge for tissue engineering is to develop a vascular supply that can support the metabolic needs of the engineered tissues in an extracellular matrix. In this study, the feasibility of using a natural compound, ginsenoside Re, isolated from Panax ginseng in stimulating angiogenesis and for tissue regeneration was evaluated. METHODS: Effects of ginsenoside Re on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were examined in vitro. Additionally, angiogenesis and tissue regeneration in a genipin-fixed porous acellular bovine pericardium (extracellular matrix; ECM) incorporated with ginsenoside Re implanted subcutaneously in a rat model were investigated. Basic fibroblast growth factor (bFGF) was used as a control. RESULTS: It was found that HUVEC proliferation, migration in a Transwell plate, and tube formation on Matrigel were all significantly enhanced in the presence of bFGF or ginsenoside Re. Additionally, effects of ginsenoside Re on HUVEC proliferation, migration, and tube formation were dose-dependent and reached a maximal level at a concentration of about 30 microg/ml. The in vivo results obtained at 1 week postoperatively showed that the density of neocapillaries and the tissue hemoglobin content in the ECMs were significantly enhanced by bFGF or ginsenoside Re. These results indicated that angiogenesis in the ECMs was significantly enhanced by loading with bFGF or ginsenoside Re. At 1 month postoperatively, vascularzied neo-connective-tissue fibrils were found to fill the pores in the ECMs loaded with bFGF or ginsenoside Re. CONCLUSIONS: The aforementioned results indicated that like bFGF, ginsenoside Re-associated induction of angiogenesis enhanced tissue regeneration, supporting the concept of therapeutic angiogenesis in tissue-engineering strategies.

Construction of varying porous structures in acellular bovine pericardia as a tissue-engineering extracellular matrix.

In the study, a cell extraction process was used to remove the cellular components from bovine pericardia. Varying pore sizes and porosities of the acellular tissues were then created using acetic acid and collagenase and subsequently fixed with genipin. Biochemical analyses found that these acellular tissues with distinct porous structures consisted primarily of insoluble collagen, elastin, and tightly bound glycosaminoglycans. The thermal stability, mechanical properties, and capability against enzymatic degradation of the bovine pericardial tissue remained unaltered after cell extraction. However, following further treatment with acetic acid and collagenase, the thermal stability and capability against enzymatic degradation of the acellular tissues declined. The porous structures of the implanted samples seem to determine whether successful microvessel-ingrowth takes place. The acetic-acid- and collagenase-treated tissues, due to their high pore size and porosity, showed a large number of microvessels infiltrating into the interstices of the implanted samples. In contrast, a low density of microvessels was observed infiltrating into the acellular tissue and penetration of microvessels into the cellular tissue was never encountered.

Acellular bovine pericardia with distinct porous structures fixed with genipin as an extracellular matrix.

A cell extraction process was employed to remove the cellular components from bovine pericardia. Various porous structures of the acellular tissues were then created, using acetic acid and collagenase, and subsequently fixed with genipin. The biological response and tissue regeneration pattern for each studied group were evaluated in a growing rat model. One month postoperatively, fibroblasts, neoconnective tissue fibrils, and neocapillaries were observed in the acellular, acetic acid-treated, and collagenase-treated tissues to fill the pores within the implanted samples, indicating that these tissue samples were being regenerated. The neoconnective tissue fibrils were identified to be neocollagen fibrils and neoglycosaminoglycans. On the other hand, no tissue regeneration was observed in the cellular tissue throughout the entire course of the study; tissue regeneration was limited to the outer most layer of the acellular tissue. In contrast, the areas of tissue regeneration in the acetic acid-treated and collagenase-treated tissues were expanded with increasing duration of implantation. However, 1 year postoperatively there were still numerous inflammatory cells observed in the acetic acid-treated tissue, whereas inflammatory cells in the collagenase-treated tissue had almost disappeared. These results indicated that tissue regeneration patterns within acellular tissues were significantly affected by their porous structures.

Tissue regeneration patterns in acellular bovine pericardia implanted in a canine model as a vascular patch.

It was noted in our previous study that acellular tissues can provide a natural microenvironment for host cell migration and proliferation to accelerate tissue regeneration. The purpose of this study was to further investigate the tissue regeneration patterns in acellular bovine pericardia fixed with glutaraldehyde or genipin as a biological patch to repair a defect in the pulmonary trunk in a canine model. The implanted samples were retrieved at distinct durations postoperatively. The structural remodeling of retrieved samples was then examined. It was found that the degree of inflammatory reaction observed for the genipin-fixed acellular patch was significantly less than its glutaraldehyde-fixed counterpart. At 1 month postoperatively, intimal thickening was found on the inner surfaces of both studied groups. The intimal thickening observed on the glutaraldehyde-fixed acellular patch was significantly thicker than its genipin-fixed counterpart. An intact layer of endothelial cells was found on the intimal thickening of the genipin-fixed acellular patch, whereas endothelial cells did not universally and totally cover the entire surface of the glutaraldehyde-fixed acellular patch. Additionally, fibroblasts with neocollagen fibrils and myofibroblasts were observed in the acellular patches for both studied groups, an indication of tissue regeneration. This phenomenon was more prominent for the genipin-fixed acellular patch than its glutaraldehyde-fixed counterpart. At 6 months postoperatively, foci of chondroid and/or bony metaplasia were found in each retrieved sample for both studied groups. The observed adverse response of chondroid metaplasia may be attributed to a compliance mismatch at the implanted site of the canine pulmonary trunk after implantation or a lack of angiogenesis in the regenerated tissue observed at 1 month postoperatively. Bony metaplasia may then develop as in other chondroid tissues. It was reported that ischemia is a usual cause of metaplasia.

Effects of crosslinking degree of an acellular biological tissue on its tissue regeneration pattern.

It was reported that acellular biological tissues can provide a natural microenvironment for host cell migration and may be used as a scaffold for tissue regeneration. To reduce antigenicity, biological tissues have to be fixed with a crosslinking agent before implantation. As a tissue-engineering scaffold, it is speculated that the crosslinking degree of an acellular tissue may affect its tissue regeneration pattern. In the study, a cell extraction process was employed to remove the cellular components from bovine pericardia. The acellular tissues then were fixed with genipin at various known concentrations to obtain varying degrees of crosslinking. It was shown in the in vitro degradation study that after fixing with genipin, the resistance against enzymatic degradation of the acellular tissue increased significantly with increasing its crosslinking degree. In the in vivo subcutaneous study, it was found that cells (inflammatory cells, fibroblasts, endothelial cells, and red blood cells) were able to infiltrate into acellular tissues. Generally, the depth of cell infiltration into the acellular tissue decreased with increasing its crosslinking degree. Infiltration of inflammatory cells was accompanied by degradation of the acellular tissue. Due to early degradation, no tissue regeneration was observed within fresh (without crosslinking) and the 30%-degree-crosslinking acellular tissues. This is because the scaffolds provided by these two samples were already completely degraded before the infiltrated cells began to secrete their own extracellular matrix. In contrast, tissue regeneration (fibroblasts, neo-collagen fibrils, and neo-capillaries) was observed for the 60%- and 95%-degree-crosslinking acellular tissues by the histological examination, immunohistological staining, transmission electron microscopy, and denaturation temperature measurement. The 95%-degree-crosslinking acellular tissue was more resistant against enzymatic degradation than its 60%-degree-crosslinking counterpart. Consequently, tissue regeneration was limited in the outer layer of the 95%-degree-crosslinking acellular tissue throughout the entire course of the study (1-year postoperatively), while tissue regeneration was observed within the entire sample for the 60%-degree-crosslinking acellular tissue. In conclusion, the crosslinking degree determines the degradation rate of the acellular tissue and its tissue regeneration pattern.

The effect of galectin 1 on 3T3 cell proliferation on chitosan membranes.

Galectin-1 (GAL1), a beta-galactoside-binding protein, functions in cell adhesion, development, and growth regulation. A number of studies suggest that GAL1 play an important role in enhancing cell adhesion to extracellular matrix and inducing cell proliferation. Chitosan is a derivative of chitin extracted from lobsters, crabs and shrimps' exoskeletons. In clinical medicine, chitosan membrane had been used as a semi-permeable biological dressing. Although chitosan membranes show no cytotoxicity, some cell types (e.g. 3T3 cells) fail to attach and proliferate on their surface. In these studies, we show that over-expression of GAL1 does not enhance 3T3 cell proliferation on chitosan membranes. However, coating the chitosan membrane with recombinant GAL1 proteins significantly expedites 3T3 cells proliferation. The enhanced cell growth was inhibited by thiodigalactoside (TDG, a potent inhibitor of beta-galactoside binding) and GAL1 monoclonal antibodies, suggesting GAL1's specific effect on the proliferation of 3T3 cells upon chitosan membranes. Moreover, immunoblotting detected a markedly suppressed tyrosine phosphorylation in several proteins on 3T3 cell growths upon GAL1-coated chitosan membrane. Pretreating the cells with sodium fluoride (NaF, a phosphatase inhibitor) inhibits the attachment and proliferation of 3T3 cells. These findings support a proposed role for altered levels of protein phosphorylation in GAL1-mediated cell attachment and proliferation on chitosan membranes.

Genipin-crosslinked gelatin microspheres as a drug carrier for intramuscular administration: in vitro and in vivo studies.

Gelatin microspheres have been widely evaluated as a drug carrier. Nevertheless, gelatin dissolves rather rapidly in aqueous environments, making the use of the polymer difficult for the production of long-term delivery systems. This adverse aspect requires the use of a crosslinking agent in forming nonsoluble networks in microspheres. However, the use of crosslinking agents such as formaldehyde and glutaraldehyde can lead to toxic side effects owing to residual crosslinkers. In an attempt to overcome this problem, a naturally occurring crosslinking agent (genipin) was used to crosslink gelatin microspheres as a biodegradable drug-delivery system for intramuscular administration. Glutaraldehyde was used as a control. In the in vitro study, the morphology, dynamic swelling, and antienzymatic degradation of test microspheres were evaluated. In the in vivo study, the biocompatibility and degradability of test microspheres were implanted in the skeletal muscle of a rat model via intramuscular injection. The results obtained in the study suggested that crosslinking of gelatin microspheres with glutaraldehyde or genipin may produce distinct crosslinking structures. The water transport mechanism in both the glutaraldehyde- and genipin-crosslinked gelatin microspheres exhibit anomalous behavior ranging from Fickian to Case-II extremes. The increase of the swelling diameter for the genipin-crosslinked microspheres was significantly less than that observed for the glutaraldehyde-crosslinked microspheres. In the animal study, it was found that the degree in inflammatory reaction for tissues implanted with the genipin-crosslinked microspheres was significantly less than that implanted with the glutaraldehyde-crosslinked microspheres. Additionally, the degradation rate of the genipin-crosslinked microspheres was significantly slower than their glutaraldehyde-crosslinked counterparts. These results indicated that the genipin-crosslinked gelatin microspheres may be used as a long-acting drug carrier for intramuscular administration.

In vivo evaluation of cellular and acellular bovine pericardia fixed with a naturally occurring crosslinking agent (genipin).

A cell extraction process was employed in the study to remove the cellular components from bovine pericardium, leaving a framework of largely insoluble collagen and elastin. It was hypothesized in the literature that this process may decrease the antigenic load (or increase the biocompatibility) within the material. Additionally, acellular tissues may provide a natural microenvironment for host-cell migration to regenerate the tissue. The study was to evaluate the biocompatibility of cellular and acellular bovine pericardia fixed with a naturally occurring crosslinking agent (genipin) implanted subcutaneously in a growing rat model. Additionally, the tissue regeneration rate in the genipin-fixed acellular tissue was investigated. The glutaraldehyde-fixed counterparts were used as controls. The results indicated that the degrees in inflammatory reaction for the genipin-fixed cellular and acellular tissues were significantly less than their glutaraldehyde-fixed counterparts. Additionally, it was noted that the inflammatory reactions for the glutaraldehyde-fixed cellular and acellular tissues lasted much longer than their genipin-fixed counterparts. The tissue regeneration rate for the genipin-fixed acellular tissue was significantly faster than its glutaraldehyde-fixed counterpart. The calcium content of each studied group, analyzed by atomic absorption. did not change significantly until at the 52nd week, postoperatively. The differences in calcium content between the cellular and acellular tissues were insignificant for both the glutaraldehyde- and genipin-fixed groups throughout the entire course of the study. In summary, the biocompatibility of the genipin-fixed cellular and acellular tissues was superior to their glutaraldehyde-fixed counterparts. The genipin-fixed acellular tissue provided a better microenvironment for tissue regeneration than its glutaraldehyde-fixed counterpart, due to its low cytotoxicity. These results suggested that the genipin-fixed acellular tissue might be used as a tissue-engineering matrix in the clinical applications.