In vitro and in vivo evaluation of chitosan-gelatin scaffolds for cartilage tissue engineering.

Chitosan-gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan-gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1WSC constructs (0.187±0.095 µg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329±0.660 µg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.

Bone marrow mesenchymal stem cells, platelet-rich plasma and nanohydroxyapatite-type I collagen beads were integral parts of biomimetic bone substitutes for bone regeneration.

Platelet rich plasma (PRP), which includes many growth factors, can activate osteoid production, collagen synthesis and cell proliferation. Nanohydroxyapatite-type I collagen beads (CIB), which mimetic natural bone components, are not only flexible fillers for bone defect but also encourage osteogenesis. Bone marrow mesenchymal stem cells (BMSCs) are often used as an abundant cell source for tissue engineering. We used a rabbit model to combine PRP, CIB and BMSCs (CIB+PRP+BMSC) into a bone-like substitute to study its impact on bone regeneration, when compared to defect alone, PRP, CIB+PRP, and PRP+BMSC. CIB+PRP upregulated more alkaline phosphatase (ALP) activity in BMSCs than PRP alone at 4 weeks postoperation. CIB+PRP+BMSC and PRP+BMSC did not differ significantly in DNA content, total collagen content, and ALP activity at 8 weeks. In histological assay, both CIB+PRP+BMSC and PRP+BMSC showed more bone regeneration at 4 and 8 weeks. Higher trabecular bone volume in tissue volume (BV/TV) (31.15±2.67% and 36.93±1.01%), fractal dimension (FD) (2.30±0.18 and 2.65±0.02) and lower trabecular separation (Tb.Sp) (2.30±0.18 and 1.35±0.16) of CIB+PRP+BMSC than of other groups at 4 and 8 weeks, and approach to of bone tissue (BV/TV=24.35±2.13%; FD=2.65±0.06; Tb.Sp=4.19±0.95). CIB+PRP+BMSC significantly enhanced new bone formation at 4 week. Therefore, nanohydroxyapatite-type I collagen beads combined with PRP and BMSCs produced a bone substitute with efficiently improved bone regeneration that shows promise to repair bone defects.

Effect of hyperbaric oxygenation on intervertebral disc degeneration: an in vivo study with sprague-dawley rats.

STUDY DESIGN: An in vivo study was conducted to test the effect of hyperbaric oxygenation (HBO) on intervertebral disc degeneration in Sprague-Dawley rats. OBJECTIVE: To observe the changes in intervertebral disc height and levels of glycosaminoglycan, collagen, interleukin-1ß (IL-1ß), prostaglandin E2 (PGE2), and inducible nitric oxide synthase (iNOS) in degenerated intervertebral discs after HBO therapy. SUMMARY OF BACKGROUND DATA: Although the involvement of IL-1ß, PGE-2, NO, and low O2 concentration has been demonstrated in intervertebral disc degeneration, the actual mechanism is not clear. It has been reported that HBO influences changes in IL-1ß, PGE-2, NO, and O2 concentration. Previously, a study demonstrated an in vitro positive effect of HBO on the human nucleus pulposus. Thus, an in vivo study in animals was necessary. METHODS: Twelve Sprague-Dawley rats were each injected with chondroitinase ABC in 2 proximal intervertebral discs of the tail. After treating with 100% oxygen at 2.5 atmospheres 2 hours per days for 10 days, the change in disc height was determined by radiography. The amounts of PGE-2, iNOS, glycosaminoglycan, and total collagen in the intervertebral disc were quantified by enzyme-linked immunosorbent assay. Tissue morphology and the distribution of glycosaminoglycan, IL-1ß, and iNOS in the intervertebral disc were assessed by histology and immunohistochemistry. The area of IL-1ß in the intervertebral discs was quantified using image analysis software. RESULTS: HBO therapy stopped the decrease in intervertebral disc height, caused an increase in the amount of glycosaminoglycan, and inhibited IL-1ß, PGE-2, and iNOS production. CONCLUSION: HBO provides a potential treatment modality for intervertebral disc degeneration.

Bioengineered periosteal progenitor cell sheets to enhance tendon-bone healing in a bone tunnel.

BACKGROUND: Tendon-bone tunnel healing is crucial for long term success in anterior cruciate ligament (ACL) reconstruction. The periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondroblasts during tendon-bone healing. We developed a scaffold-free method using polymerized fibrin-coated dishes to make functional periosteal progenitor cell (PPC) sheets. Bioengineered PPC sheets for enhancing tendon-bone healing were evaluated in an extra-articular bone tunnel model in rabbit. METHODS: PPC derived from rabbit tibia periosteum, cultivated on polymerized fibrin-coated dishes and harvested as PPC sheet. A confocal microscopy assay was used to evaluate the morphology of PPC sheets. PPC sheets as a periosteum to wrap around hamstring tendon grafts were pulled into a 3-mm diameter bone tunnel of tibia, and compared with a tendon graft without PPC sheets treatment. Rabbits were sacrificed at 4 and 8 weeks postoperatively for biochemical as-say and histological assay to demonstrate the enhancement of PPC sheets in tendon-bone healing. RESULTS: PPC spread deposit on fibrin on the dish surface with continuous monolayer PPC was ob-served. Histological staining revealed that PPC sheets enhance collagen and glycosaminoglycans deposition with fibrocartilage formation in the tendon-bone junction at 4 weeks. Collagen fiber with fibrocartilage formation at tendon-bone junction was also found at 8 weeks. Matured fibrocartilage and dense collagen fiber were formed at the tendon-bone interface at 8 weeks by Masson trichrome and Safranin-O staining. CONCLUSIONS: Periosteal progenitor cell monolayer maintains the differentiated capacity and osteochondral potential in order to promote fibrocartilage formation in tendon-bone junction. Bioengineered PPC sheets can offer a new feasible therapeutic strategy of a novel approach to enhance tendon-bone junction healing.

Enhancement of rotator cuff tendon-bone healing with injectable periosteum progenitor cells-BMP-2 hydrogel in vivo.

PURPOSE: The fixation and incorporation of ruptured rotator cuff tendon to bone is a major concern in rotator cuff repair surgery. Rotator cuff repair usually fails at the tendon-bone interface, especially in case of large or massive tears. To enhance tendon-bone healing, an injectable hydrogel made with periosteal progenitor cells(PPCs) and poly (ethylene glycol) diacrylate (PEGDA) tethered with bone morphogenic protein-2(BMP-2) was developed to encourage extracellular matrix synthesis for tendon-to-bone healing in rotator cuff repair. METHODS: The infraspinatus tendon was cut from the greater tuberosity and repaired through a transosseous tunnel with the injectable progenitor cell-BMP-2 hydrogel applied between the tendon-bone interface. The injectable hydrogel was prepared from 10% poly (ethylene glycol) diacrylate (PEGDA) containing 0.05% of the photoinitiator. BMP-2 tethered with poly(ethylene glycol) (PEG) was blended to the hydrogel. Rabbit periosteal progenitor cells (PPCs) isolated from periosteum were mixed with hydrogel and injected on the tendon-bone interface. Ultraviolet radiation (365 nm) was applied for 60 s to photopolymerize the injection and solidify the hydrogel. The rabbits were killed at 4 and 8 weeks. The morphological characteristics of the healing tendon-to-bone interface were evaluated by histological and immunohistochemical methods. The biomechanical test was done to determine healing attachment strength. RESULTS: At both the 4- and 8-week killing, histological analysis of the tendon-bone interface showed an increasing fibrocartilage and bone layer formed in the tendon-bone interface in PEGDA group. At 4 weeks, fibrocartilage-like tissue was observed in a focal area. At 8 weeks, further matrix deposition occurred with fibrocartilage formation in the tendon-bone junction, and bone formation appeared near host bone. Immunohistochemistry revealed the presence of aggrecan and type II collagen. Biomechanical testing revealed a higher maximum pull-out load at all time points with a statistically significant difference at 4 and 8 weeks postoperatively. CONCLUSION: PEGDA hydrogel was approved as an adequate matrix for the encapsulation of cells and signal factor, and as an effective local delivery method to the tendon-bone interface through injection and photopolymerization. The PPCs-BMP2-hydrogel provides a powerful inductive ability between the tendon and the bone and enhances tendon-bone healing through the neoformation of fibrocartilage.

The effect of ultrasound stimulation versus bioreactors on neocartilage formation in tissue engineering scaffolds seeded with human chondrocytes in vitro.

Pulsed ultrasound (1 MHz, 67 mW/cm(2) Ispta, and 10 min/day) promoted cell proliferation and matrix deposition in low-density 2D ( approximately 6 x 10(3)cells/cm(2)) as well as 3D ( approximately 4 x 10(6)cells/cm(3)) chondrocyte cultures. The beneficial effect of ultrasound on neocartilage formation only last 28 days, shorter than that of bioreactors.

Evaluation of biodegradable polyesters modified by type II collagen and Arg-Gly-Asp as tissue engineering scaffolding materials for cartilage regeneration.

Synthetic biodegradable polyesters poly(L-lactide) (PLLA) and poly(D,L-lactide-coglycolide) (PLGA) (50:50) modified by porcine type II collagen and an Arg-Gly-Asp (RGD)-containing protein were evaluated as scaffolds for cartilage regeneration in this study. Cytocompatibility of the polymer films was tested using immortalized chondrocytes. Neocartilage formation in vitro on cell-seeded scaffolds was further examined using primary porcine chondrocytes. The inflammatory response of the scaffolds was evaluated subcutaneously in rats. A pilot animal study was conducted, in which rabbit allogeneic chondrocyte-seeded scaffolds were implanted to repair the defected rabbit knee cartilage. The results demonstrated that PLGA as well as its blends with PLLA had better cell growth than pure PLLA, and that type II collagen enhanced, but RGD inhibited cell proliferation. Scaffolds made of blended PLLA/PLGA had larger dynamic compressive modulus compared to scaffolds made of PLLA or PLGA single polymer. Chondrocyte-seeded scaffolds modified by type II collagen without RGD had the greater number of cells as well as higher glycosaminoglycan (GAG) and collagen contents compared to scaffolds without type II collagen modification or scaffolds further modified with RDG. Type II collagen modification prevented infiltration by host tissue and capsule formation. Unmodified PLLA and PLLA/PLGA constructs demonstrated persisting inflammatory response after 6 months, while all type II collagen-modified PLLA/PLGA constructs showed complete repair and no inflammation. Partial or full repair was observed after 2 months of postimplantation in type II collagen-modified PLLA/PLGA constructs, with equal cellularity and 75-80% matrix contents of a normal rabbit articular cartilage. It was concluded that PLLA/PLGA blended scaffolds modified by type II collagen were a potential tissue engineering scaffold for cartilage regeneration.

The effect of two different bioreactors on the neocartilage formation in type II collagen modified polyester scaffolds seeded with chondrocytes.

The effect of dynamic culture conditions on neocartilage formation in type II collagen modified polyester scaffolds was studied. Porcine or human articular chondrocytes were seeded in the scaffolds. The cell-scaffold constructs were cultivated statically, in a rotating-type bioreactor or in a shaker for up to 4 weeks. The cell proliferation, morphology, NO production, synthesis of proteoglycans and collagen, and mechanical properties were evaluated. The results demonstrated that the rotating-type bioreactor promoted the growth of primary porcine chondrocytes, helped to maintain their phenotype, and increased the production of extracellular matrix. The constructs also had the largest dynamic compressive modulus. In the static condition, chondrocytes occupied only the outer margin of the cell-polymer constructs. The poor mass transfer in static condition may have caused a lower pH value in the middle of the constructs and lead further to faster scaffold degradation as well as the weakest neocartilage. Constructs in the shaker produced the highest amount of NO as well as the lowest amount of cells and matrix production. Human or porcine chondrocytes of the second passage seeded in scaffolds were much less viable, with the largest amount of cells and matrix when cultured in rotating-type bioreactors. A larger seeding density was required to form neocartilage from passaged adult chondrocytes.

Evaluation of chitosan-alginate-hyaluronate complexes modified by an RGD-containing protein as tissue-engineering scaffolds for cartilage regeneration.

In this study, a series of natural biodegradable materials in the form of chitosan (C)-alginate (A)-hyaluronate (H) complexes are evaluated as tissue-engineering scaffolds. The weight ratio of C/A is 1 : 1 or 1 : 2. Sodium hyaluronate is mixed in 2%. The complexes can be cast into films or fabricated as scaffolds. Their surface can be further modified by an Arg-Gly-Asp (RGD)-containing protein, a cellulose-binding domain-RGD (R). Cytocompatibility tests of the films are conducted using immortalized rat chondrocyte (IRC) as well as primary articular chondrocytes harvested from rabbits. The neocartilage formation in cell-seeded scaffolds is examined in vitro as well as in rabbits, where the scaffolds are implanted into the defect-containing joints. The results from cytocompatibility tests demonstrate that R enhances cell attachment and proliferation on C-A and C-A-H complex films. Complex C1A1HR (C : A = 1 : 1 with H and R) has better performance than the other formulation. Cells retain their spherical morphology on all C-A and C-A-H complexes. The in vitro evaluation of the seeded scaffolds indicates that the C1A1HR complex is the most appropriate for 3-D culture, manifested by the better cell growth as well as higher glycosaminoglycan and collagen contents. When the chondrocyte scaffolds are implanted into rabbit knee cartilage defects, partial repair is observed after 1 month in C1A1HR as well as in C1A1 (C : A = 1 : 1 without H and R) scaffolds. The defects are completely repaired in 6 months when C1A1HR constructs are implanted. It is concluded that C1A1HR is a potential tissue-engineering scaffold for cartilage regeneration.