In vivo angiogenesis effect of porous collagen scaffold with hyaluronic acid oligosaccharides.

BACKGROUND: Tissue engineering is a promising solution for tissue defect repair. A key problem, however, is how to keep the engineered tissue alive after implantation. The ideal scaffold for tissue engineering would be biocompatible and biodegradable and, more importantly, would exhibit good interaction with endothelial cells to promote angiogenesis. MATERIALS AND METHODS: Three different scaffolds were synthesized: collagen/hyaluronic acid (HA) (MW 6.5K), collagen/HA (MW 220K), and collagen only. The synthesized collagen/HA scaffold was analyzed for water content, pore size, and HA content. An animal model for in vivo tissue construct angiogenesis was developed using the inferior epigastric skin flap of mice and perfusion of quantum dots; the average fluorescence intensity per unit area was calculated and correlated with vessel density from histologic examination. RESULTS: The pore size is not statistically different among the three groups and the HA content is not statistically different between the two collagen/HA groups. The fluorescence intensity of the collagen/HA (MW 6.5K) group is increased at day 14, 21, and 28, and is significantly higher than in the other groups. Similar results were also obtained from histologic immunohistochemistry studies. CD31-stained vessels were found co-localized with QD fluorescence and these newly formed vessels were identified at day 14 in the collagen/HA (MW 6.5K) group and increased significantly at day 21 and 28. CONCLUSION: This study showed that collagen scaffolds with short-chain HA (MW 6.5K) revascularize faster than those with long-chain HA (MW 220K) and collagen only. The results of the new animal model for studying scaffold angiogenesis are compatible with the conventional methods of immunostaining and histological examination.

Fibronectin and laminin promote differentiation of human mesenchymal stem cells into insulin producing cells through activating Akt and ERK.

BACKGROUND: Islet transplantation provides a promising cure for Type 1 diabetes; however it is limited by a shortage of pancreas donors. Bone marrow-derived multipotent mesenchymal stem cells (MSCs) offer renewable cells for generating insulin-producing cells (IPCs). METHODS: We used a four-stage differentiation protocol, containing neuronal differentiation and IPC-conversion stages, and combined with pellet suspension culture to induce IPC differentiation. RESULTS: Here, we report adding extracellular matrix proteins (ECM) such as fibronectin (FN) or laminin (LAM) enhances pancreatic differentiation with increases in insulin and Glut2 gene expressions, proinsulin and insulin protein levels, and insulin release in response to elevated glucose concentration. Adding FN or LAM induced activation of Akt and ERK. Blocking Akt or ERK by adding LY294002 (PI3K specific inhibitor), PD98059 (MEK specific inhibitor) or knocking down Akt or ERK failed to abrogate FN or LAM-induced enhancement of IPC differentiation. Only blocking both of Akt and ERK or knocking down Akt and ERK inhibited the enhancement of IPC differentiation by adding ECM. CONCLUSIONS: These data prove IPC differentiation by MSCs can be modulated by adding ECM, and these stimulatory effects were mediated through activation of Akt and ERK pathways.

Fabrication of vascularized bone grafts of predetermined shape with hydroxyapatite-collagen gel beads and autogenous mesenchymal stem cell composites.

BACKGROUND: Advances in tissue-engineering techniques have enabled new procedures to be developed for bone regeneration. In this study, for engineering of structural tissues with supporting vascular networks, the authors attempted to produce vascularized tissue-engineered bone grafts using cultured mesenchymal stem cells/hydroxyapatite/collagen gel bead composites and vascular bundle implantation. METHODS: Twenty-four New Zealand White rabbits underwent implantation of ringed polytetrafluoroethylene vascular grafts (1 x 3 cm) in the medial thigh with the femoral vascular bundle passing through. The polytetrafluoroethylene grafts were left unfilled (group A), filled with hydroxyapatite/collagen gel beads (group B), or filled with mesenchymal stem cells/hydroxyapatite/collagen gel bead composites (group C). At 4, 8, 12, and 16 weeks, the implants were removed and radiographic and histologic examinations were conducted. RESULTS: Radiographic analysis revealed that the area of radiopacity within the chamber was highest in group C. The average calcified densities of groups B and C were between 0.99 +/- 0.11 and 1.29 +/- 0.14. Histologically, there was fibroadipose tissue within the chamber in group A. New tissue had grown into the matrix of the chambers of groups B and C, and substitution of the biomaterials was seen. Newly formed fibrovascular networks and osteoids were simultaneously seen. Bone marrow was observed in the vascular graft of group C 6 months after implantation. CONCLUSIONS: Tissue-engineered vascularized bone grafts of predetermined shape were created with mesenchymal stem cell/hydroxyapatite/collagen gel bead composites. The results of this study showed that successful in vivo engineering of vascularized tissue-engineered bone grafts is possible.

Type I collagen promotes proliferation and osteogenesis of human mesenchymal stem cells via activation of ERK and Akt pathways.

Biomaterials not only serve as scaffolds for bone regeneration, but may also exhibit inductive capability for bone growth. The goal of this study was to identify the best extracellular matrix protein for enhancing osteogenesis by hMSCs (human mesenchymal stem cells) and to investigate the underlying mechanism. Coating with collagen I, but not fibronectin, laminin, gelatin, and poly-L-lysine, enhanced late cell proliferation and promoted osteogenesis by hMSCs, as evidenced by an increase in Alizarin Red S staining, alkaline phosphatase activity and mRNA levels of Runx2 and osteocalcin. Coating with collagen I induced activation of ERK and Akt but not FAK, and treatment with PD98059 and LY294002 blocked the activation of ERK and Akt, respectively. Interestingly, LY294002 also blocked ERK activation, indicating the activation of PI3K/ERK pathway upon contact with collagen I. Furthermore, PD98059 or LY294002 abolished collagen I-induced promotion of osteogenesis by hMSCs. However, blocking antibodies against alpha2beta1 integrins did not inhibit collagen I-induced osteogenesis by hMSCs. These data demonstrate that collagen I promotes proliferation and osteogenesis of hMSCs via activation of ERK and Akt pathways.

Apoptosis in chondrogenesis of human mesenchymal stem cells: effect of serum and medium supplements.

Apoptosis is an inevitable process during development and is evident in the formation of articular cartilage and endochondral ossification of growth plate. Mesenchymal stem cells (MSCs) can serve as alternative sources for cell therapy in focal chondral lesions or diffuse osteoarthritis. But there are few, if any, studies investigating apoptosis during chondrogenesis by MSCs. The aim of this study was to find the better condition to prevent apoptosis during chondrogenesis by MSCs. Apoptosis were evaluated in MSCs induced in different chondrogenic media by the use of Annexin V, TUNEL staining, lysosomal labeling with lysotracker and immunostaining of apoptotic markers. We found apparent apoptosis was demonstrated by Annexin V, TUNEL staining and lysosomal labeling during chondrogenesis. Meanwhile, the degree of apoptosis was related to the reagents of the defined chondrogenic medium. Adding serum in medium increased apoptosis, however, TGF-beta1 inhibited apoptosis. The apoptosis was associated with the activation of caspase-3, the increase in the Bax/Bcl-2 ratio, the loss of lysosomal integrity, and the increase of PARP-cleavage. Pro-inflammatory cytokines, IL-1alpha, IL-1beta and TNFalpha did not induce any increase in apoptosis. Interestingly, the inhibition of apoptosis by serum free medium supplemented with ITS was also associated with an increase in the expression of type II collagen, and a decrease in the expression of type X collagen, Runx2, and other osteogenic genes, while TGF-beta1 increased the expression of Sox9, type II and type X collagen and decreased the expression of osteogenic genes. These data suggest apoptosis occurs during chondrogenesis by MSCs by cell death intrinsic pathway activation and this process may be modulated by culture conditions.

The effects of fiber size on MG63 cells cultured with collagen based matrices.

The behavior of human osteoblast-like MG63 cells cultured on electrospun collagen fibers of three different sizes (50-200 nm, 200-500 nm, and 500-1000 nm in diameter) were investigated. The growth of MG63 cells on all three electrospun collagen fibers are the same and about 70% higher than those cultured on monomeric collagen and tissue-culture polystyrene (TCPS). The migration speed of MG63 cells, on the other hand, decreased as the diameter of nanofibers increased. There were more distinct actin stress fibers formed in MG63 cells when the cells cultured on collagen substrates as compared with TCPS. In addition, MG63 cells displayed different adhesion and spreading patterns on different sizes of collagen fibers. Size variation of collagen nanofibers apparently has more impact on cell migration distance and cell morphology as compared with cell growth. It was demonstrated that collagen nanofibers promoted MG63 cell interaction with matrices by providing a suitably rough nanometer surface. The results of this study present important information for the development of collagen-based biomaterials.

Fabrication of pre-determined shape of bone segment with collagen-hydroxyapatite scaffold and autogenous platelet-rich plasma.

BACKGROUND: Reconstruction of large segment of bony defects is frequently needed in hand surgery. Autogenous bone grafting is considered the standard in management of these bony defects but has limited source of graft material. Collagen and hydroxyapatite have been used as bone-filling materials and are known to serve as the osteoconductive scaffold for bone regeneration. On the other hand, platelet-rich plasma is a kind of natural source of growth factors, and has been used successfully in bone regeneration and improving wound healing. This study was designed to evaluate the effectiveness of using biohybrids of platelet-rich plasma and collagen-hydroxyapatite beads for fabricating of protrusive bone in a rabbit animal model. METHODS: Biomaterial beads comprised of particulate hydroxyapatite dispersed in fibrous collagen (type I) matrices were prepared and filled in the ringed polytetrafluoroethylene (PTFE) artificial vascular graft (3 cm long, 1 cm in diameter). New Zealand White rabbits were each implanted with two cylindrical PTFE artificial vascular graft over both iliac crests (n = 16). A 2 x 0.5 cm opening was created at the side of each PTFE chamber to allow the content of chamber in contact with the bone marrow of the ileum. The chambers were empty (groups A and D), filled with type I collagen/hydroxyapatite beads (groups B and C). In groups C and D, autologous platelet rich plasma (PRP) was given by transcutaneous injection method into the chambers every week. After 12 weeks, the animals were sacrificed and the chambers were harvested for radiological and histological analysis. RESULTS: In plain radiographs, the group C chambers had significantly higher bone tissue engineered (average calcified density 0.95, average calcified area 61.83%) compared with other groups (P < 0.001). In histological examination, there was a creeping substitution of the implant by the in-growth of fibrovascular tissue in group C. Abundant fibrovascular networks positioned interstitially between these biomaterial beads in all parts of chamber. Degradation of these beads and newly formed capillaries and osteoids around the degraded matrixes are shown. The continually calcification in the matrixes or degraded matrixes is evidenced by the strong green fluorescence observed under the confocal microscope. In group B, looser architecture without evidence of tissue in-growth was shown. In the scaffold absent groups (A and D), there was only fibrous tissue shown within the chamber. CONCLUSIONS: In this study, we have demonstrated a feasible approach to fabricate an osseous tissue that meets clinical need. Using the type I collagen/ hydroxyapatite gel beads matrixes and intermittent injection of autologous platelet-rich-plasma, specific 3D osseous tissues with fibrovascular network structure from pre-exist bony margin were successfully created in an in vivo animal model.

Characterization and osteogenic effects of mesenchymal stem cells on microbeads composed of hydroxyapatite nanoparticles/reconstituted collagen.

Natural bone is comprised of nanosized blade-like crystals of hydroxyapatite grown in close contact with collagen (Col) fibers. Characteristics of artificial bone tissue differ considerably with those of natural ones, mainly from the unusual self-organizing interaction between the apatite crystals and the proteic components. Nanoparticle spheres of hydroxyapatite (n-HA), dispersed in reconstituted fibrous Col, were prepared in three weight ratios of 75:25, 65:35, and 50:50 (n-HA:Col). Bone marrow mesenchymal stem cells (MSCs) from rabbits were seeded and cultured on the n-HA/Col microbeads and characterized. n-HA were evenly distributed throughout the Col matrix and aggregated to microbeads as determined by scanning electron microscopy. Electron and confocal microscopy showed that the MSCs spread and attached to microbeads via focal adhesions, while staining for F-actin and DNA revealed the presence of stress fibers. The phenotype of the MSCs in the flow cytometry was identified as CD11a-, CD44+, and CD90.1+. The optimal weight ratio is 65:35 for the normalized alkaline phosphatase activities. The transduced MSCs, engineered by replication-defective adenovirus to express the BMP-2 gene, demonstrated synergic osteogenic effects in the microbeads. MSCs are capable of proliferating and differentiating in appropriate combinations of n-HA/Col. Thus it is a promising composite for future clinical applications.

Development of two alginate-based wound dressings.

Two types of new alginate-based wound dressings, Type-AP and Type-AE, were fabricated by the EDC-activated crosslinking of alginate with Polyethyleneimine and Ethylenediamine, respectively. As compared with the commercial non-woven wound dressing, Kaltostat, both Type-AP and Type-AE dressings had higher degradation temperature, lower calcium content, and a sponge-like macroporous structure. In addition, these two alginate-based dressings had higher mechanical stress (12.37 +/- 1.72 and 6.87 +/- 0.5 MPa for Type-AP and -AE, respectively) and higher water vapor transmission rates (both about 3,500 g/m2/day) than Kaltostat (0.87 +/- 0.12 MPa and 2,538 g/m2/day). Fibroblasts proliferated faster on these two newly developed wound dressings at a higher rate as compared with that on Kalostat dressing. The results of animal study showed that the wounds treated with either Type-AP or Type-AE dressings healed faster than Kaltostat with less encapsulation of residuals by fibrous tissue and more neo-capillary formation. These two newly developed Type-AP and Type-AE porous wound dressings thus have great potential for clinical applications.

Influences of hyaluronan on type II collagen fibrillogenesis in vitro.

The effect to the kinetics of type II collagen fibrillogenesis with the addition of hyaluronan (HA), (Mw of 1.8x10(6) Da), at various concentrations of HA (0.01, 0.05 and 0.1 wt.%) for a series of fibril formation systems was examined in this study. Evidences deduced from the turbidity-time curves revealed that the inclusion of HA had minor or no impact to the fibrillogenesis of type II collagen (collagen conc. at 0.2 mg/mL). The apparent rate constants, klag (lag phase) increased slightly but kg (growth phase) decreased not very significantly with addition of HA, as compared to the case of pure collagen. This leads us to believe tentatively that, with the addition of HA to collagen solutions, the nucleation process of the fibril formation might have been sped up slightly whereas the growth process slowed up slightly. However, data from TEM observations on the resulting fibrils indicated that the presence of HA did not significantly affect the diameters and the characteristic D-banding periods of the collagen fiber formed. And, from the statistical analyses, we found only insignificant difference (P>0.05) between the specimens from the various experimental groups. It seems to indicate that the ultimate packing of collagen monomers was probably not interfered or affected significantly by the presence of HA in vitro.

Microspheres of collagen/beta-TCP with an open network fibrillar structure strengthened by chitosan.

A novel method of preparing collagen/beta-tricalcium phosphate microspheres with chitosan as the mechanical strength enhancer has been developed in this study. The process involved firstly droplet formation by discharging a mixture of collagen, beta-tricalcium phosphates and alginate into an aqueous solution of CaCl(2) by extruding through an air jet-syringe at 4 degrees C. The gel beads thus formed were collected and subsequently coated with chitosan to stabilize the surface of gel bead. Collagen within the gel beads was then reconstituted while the entrapped alginate was liquefied and drained by incubating in phosphate buffer at 37 degrees C. Microspheres comprised of fibrillar collagen and well-dispersed beta-tricalcium phosphate particulates were obtained by this process. And the mechanical strength of these microspheres was significantly enhanced by chitosan coating. These chitosan-coated collagen/beta-tricalcium phosphate microspheres have an open fibrillar network structure with a great potential for future application as biodegradable bone grafting materials.

A novel method of encapsulating and cultivating adherent mammalian cells within collagen microcarriers.

A novel method of preparing collagen microcarriers was developed and used to entrap adherent cells for cell culturing. This new technique involved seeding of cells in micro gel beads comprised of collagen fibrils dispersed in alginate. The gel beads were washed with phosphate buffered saline (PBS) to remove alginate and the resulting microspheres, about 300-500 microm in diameter, contained evenly distributed collagen fibrils which provided a 3D biomimetic environment for cell growth. The applicability of this microencapsulating system was demonstrated by its ability to support the growth of C2C12 myoblast cells. When seeded and cultured within the 3D collagen microcarriers, the population of C2C12 cells entrapped within the microcarriers increased by 1.5 folds in 7 days after inoculation. This encapsulation technique is potentially useful for culturing cells and especially useful for adherent cells that require a 3D fibrillar collagen environment.

Three-dimensional collagen fiber remodeling by mesenchymal stem cells requires the integrin-matrix interaction.

Tissue engineering aiming to repair or regenerate damaged tissues necessitates fabricating three-dimensional biomaterial scaffolds with controlled porosity for delivering cells. To facilitate cell distribution, a strategy using stem cell-based fabrication of biomaterials was tested in type II collagen fibers. Human mesenchymal stem cells when delivered in type II collagen assembled and reorganized these matrices and differentiated into spherical chondrocytes with the synthesis of cartilage proteins. The cell-mediated assembly and reorganization of collagen fibers was not limitless and only restricted to an appropriate ratio of cell number and collagen amount. The blocking of alpha2 or beta1-integrin function with specific antibodies significantly impeded the collagen-assembly effects. In vitro chondrogenesis or in vivo cartilage formation of human mesenchymal stem cells was also dependent on the interactions between cells and surrounding matrices. This method for three-dimensional fabricating collagen fibers may generally be applied to other biomaterials, when combined with surface modification or ligand addition for cell adhesion.

Growth of mesenchymal stem cells on electrospun type I collagen nanofibers.

We reconstituted type I collagen nanofibers prepared by electrospin technology and examined the morphology, growth, adhesion, cell motility, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) on three nano-sized diameters (50-200, 200-500, and 500-1,000 nm). Results from scanning electron microscopy showed that cells on the nanofibers had a more polygonal and flattened cell morphology. MTS (3-[4,5-dimethythiazol-2-yl]-5-[3-carboxy-methoxyphenyl]-2-[4-sul-fophenyl]-2H-tetrazolium compound) assay demonstrated that the MSCs grown on 500-1,000-nm nanofibers had significantly higher cell viability than the tissue culture polystyrene control. A decreased amount of focal adhesion formation was apparent in which quantifiable staining area of the cytoplasmic protein vinculin for the 200-500-nm nanofibers was 39% less compared with control, whereas the area of quantifiable vinculin staining was 45% less for both the 200-500-nm and 500-1,000-nm nanofibers. The distances of cell migration were quantified on green fluorescent protein-nucleofected cells and was 56.7%, 37.3%, and 46.3% for 50-200, 200-500, and 500-1,000 nm, respectively, compared with those on the control. Alkaline phosphatase activity demonstrated no differences after 12 days of osteogenic differentiation, and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed comparable osteogenic gene expression of osteocalcin, osteonectin, and ostepontin between cells differentiated on polystyrene and nanofiber surfaces. Moreover, single-cell RT-PCR of type I collagen gene expression demonstrated higher expression on cells seeded on the nanofibers. Therefore, type I collagen nanofibers support the growth of MSCs without compromising their osteogenic differentiation capability and can be used as a scaffold for bone tissue engineering to facilitate intramembranous bone formation. Further efforts are necessary to enhance their biomimetic properties.

The internalized CdSe/ZnS quantum dots impair the chondrogenesis of bone marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are capable of differentiating into multiple cell lineages and are useful for therapeutic applications. Labeling the MSCs with fluorescent probes is beneficial in tracing the fate of MSCs after implantation. We have introduced the CdSe/ZnS quantum dots (QDs) into the human bone marrow MSCs and examined the effects of QDs on the proliferation and chondrogenesis of the cells. The internalized QDs were found localized in perinuclear regions and remained there after a number of cell passages. The presence of QDs did not affect the proliferation of cells or the size of chondrospheres formed, when subjected to chondrogenesis induction. However, the expression of mRNA and protein of type II collagen and aggrecan in the chondrospheres was significantly inhibited in cells labeled with QDs, suggesting impaired chondrogenesis. Our results that the presence of QDs interferes with the chondrogenic differentiation of MSCs raise concerns in using the QDs as fluorescence tracers for stem cells.

The inhibition of osteogenesis with human bone marrow mesenchymal stem cells by CdSe/ZnS quantum dot labels.

CdSe/ZnS quantum dots (QDs) have recently been used as cell tracers for long term imaging of live cells. A number of studies indicate that introduction of quantum dots to cells have no apparent deleterious effects on the morphology or growth of cells. In the present study, the human bone marrow mesenchymal stem cells (hBMSCs) were used as a model to examine the effects of QDs on the growth and osteogenic differentiation of the cells. The CdSe/ZnS QDs were delivered into hBMSCs by liposome-mediated transfection with high efficiency; analysis by transmission electron microscopy revealed that the internalized QDs could be located in the endosome-like vesicles. Uptake of QDs into hBMSCs did not affect the proliferation and cell cycle distribution of the cells. When induced to differentiate along the osteogenic lineage, the QD-containing-hBMSCs were shown to have mineral deposition on the extracellular matrix. However, the cells displayed lower alkaline phosphatase activity as compared to those without QDs. Analysis by reverse transcriptase polymerase chain reaction further demonstrated that the expression of two osteogenic markers, osteopontin and osteocalcin, was significantly inhibited. Together our results show that the presence of QDs in hBMSCs prevents the full response of the cells to induced osteogenic differentiation.

Influence of alginate on type II collagen fibrillogenesis.

Collagen II is the majority of extracellular matrix components in articular cartilage, which with the major functions of preventing expansion of the tissue and distributing the load of body weight. To obtain man-made ECM, the reconstitution of collagen could be conducted in the presence of negatively charged polysaccharide, such as alginate. Alginate is an anionic polysaccharide capable of eversible gelated in calcium ion solution to prepare different shapes of biomaterials. Its well-known biocompatibility makes it an ideal material in biomedical applications. Thus, the aim of this study was to evaluate the effects of alginate on the fibrillogenesis of type II collagen. The preliminary results revealed that inclusion of alginate into soluble type II collagen solution could inhibit the development of turbidity of collagen solution, and the apparent rate constants in lag and growth phases decreased during collagen formation period, both rate constants decreased to about one-third of the original constants, respectively. From TEM observations, the collagen fibrils were significantly thicker in 0.05% and 0.1% alginate as compared with pure collagen solution. Furthermore, the D-periods of collagen fibers kept unchanged significantly under all reconstituted conditions, which meant the packing of collagen monomer was probably not affected by adding these amounts of alginate.

An in vivo study of a bone grafting material consisting of hydroxyapatite and reconstituted collagen.

This study aims to evaluate the performance of our recently developed microspheres of hydroxyapatite/reconstituted collagen as a bone grafting material. The microspheres were fabricated into a circular disc and implanted in a pre-drilled hole in a rat's calvaria. The bone tissue had regenerated and grown into the disc bone graft 4 weeks following implantation. After 16 weeks of implantation, the regenerated bone had integrated with the remaining material and made close contact with it. The disc had been completely absorbed with almost no visible bone graft left after 24 weeks of implantation. In contrast, a hydroxyapatite disc still remained intact on the 24th week after implantation. These results suggested that the hydroxyapatite/reconstituted collagen microsphere can be used as an excellent bone grafting material.

A new anti-adhesion film synthesized from polygalacturonic acid with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinker.

The most commonly used anti-adhesion device for separation and isolation of wounded tissues after surgery is the polymeric film. In this study, a new anti-adhesion membrane based on polygalacturonic acid (PGA) has been synthesized, and its biocompatibility and anti-adhesion capabilities evaluated. The PGA film was reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to obtain a cross-linked PGA film with an 86% gel content and a 47% water content when immersed in aqueous solution. This PGA-EDC film did not show any evidence of cytotoxic effects since it did not induce any significant increase in cytoplasmic LDH release from the L929 cells in contact with it. When implanted into rats, the PGA-EDC film exhibited a most promising anti-adhesion potential with only 1 out of 21 rats operated not forming any tissue adhesion. This anti-adhesion potency is significantly higher than that found for Seprafilm and untreated rats where 11 out of 21 and 18 out of 21 operated rats, respectively, formed tissue adhesions. The implanted PGA-EDC film did not elicit any acute inflammatory reaction based on the results of histological examination and peritoneal fluid leukocytes analysis. The newly developed PGA-EDC film thus has a great potential for future use in clinical applications.

Microencapsulation of parathyroid tissue with photosensitive poly(L-lysine) and short chain alginate-co-MPEG.

Human parathyroid glands were encapsulated using the alginate-PLL system in this study. In order to improve the mechanical strength and the biocompatibility, the microcapsules were fabricated with a three-layer structure that consisted of alginate/photosensitive poly(L-lysine)/short chain alginate-co-MPEG. These modified microcapsules were used for encapsulating human parathyroid tissue. In vitro experiments revealed that microencapsulated parathyroid glands maintained differentiative properties in culture, and the capsular membrane was freely permeable to the human parathyroid hormone. For in vivo experiments, these capsules were transplanted into parathyroidectomized SD-rats. After parathyroidectomy, serum calcium decreased from 2.25 to 1.68 mmol/L and remained in a constantly low concentration until transplantation. Parathyroidectomized SD-rats were normocalcemic after transplant of encapsulated parathyroid tissue. The microcapsules were then explanted at 12 weeks for examination. Histological evaluations of excised transplants revealed that the microcapsules remained intact structurally and were free of cell adhesions. The results demonstrated that human parathyroid tissue microencapsulated by this system retains stability and is functional both in vitro and in vivo. This encapsulating system will have valuable application for endocrine surgery in the future.