Platelet-rich plasma (PRP) is a fraction of plasma that contains high levels of multiple growth factors. The purpose of this study was to examine the effects of PRP on cell proliferation and matrix ...synthesis by porcine chondrocytes cultured in alginate beads, conditions that promote the retention of the chondrocytic phenotype, in order to determine the plausibility of using this plasma-derived material for engineering cartilage.
PRP and platelet-poor plasma (PPP) were prepared from adult porcine blood. Adult porcine chondrocytes were cultured in the presence of 10% PRP, 10% PPP or 10% fetal bovine serum (FBS) for 3 days. Cell proliferation, proteoglycan (PG) and collagen synthesis were quantified, and the structure of newly synthesized PG and collagen was characterized.
Treatment with 10% PRP resulted in a small but significant increase in DNA content (+11%, vs FBS;
P
<
0.01; vs PPP;
P
<
0.001). PG and collagen syntheses by the PRP-treated chondrocytes were markedly higher than those by chondrocytes treated by FBS or PPP (PG; PRP: +115% vs FBS; +151% vs PPP, both
P
<
0.0001, collagen; PRP: +163% vs FBS; +163% vs PPP, both
P
<
0.0001). Biochemical analyses revealed that treatment with PRP growth factors did not markedly affect the types of PGs and collagens produced by porcine chondrocytes, suggesting that the cells remained phenotypically stable in the presence of PRP.
PRP isolated from autologous blood may be useful as a source of anabolic growth factors for stimulating chondrocytes to engineer cartilage tissue.
Currently, functional treatment of fracture non-unions and bone loss remains a significant challenge in the field of orthopaedic surgery. Tissue engineering of bone has emerged as a new treatment ...alternative in bone repair and regeneration. Our approach is to combine a polymeric matrix with a cellular vehicle for delivery of bone morphogenetic protein-2 (BMP-2), constructed through retroviral gene transfer. The objective of this study is to develop an osteoinductive, tissue-engineered bone replacement system by culturing BMP-2-producing cells on an osteoconductive, biodegradable, polymeric-ceramic matrix. The hypothesis is that retroviral gene transfer can be used effectively in combination with a biodegradable matrix to promote bone formation. First, we examined the in vitro attachment and growth of transfected BMP-producing cells on a PLAGA–HA scaffold. Second, the bioactivity of the produced BMP in vitro was evaluated using a mouse model. It was found that the polymer-ceramic scaffold supported BMP-2 production, allowing the attachment and growth of retroviral transfected, BMP-2-producing cells. In vivo, the scaffold successfully functioned as a delivery vehicle for bioactive BMP-2, as it induced heterotopic bone formation in a SCID mouse model.
The limitations of current grafting materials have driven the search for synthetic alternatives for the regeneration of trabecular bone. A variety of biodegradable polymer foams composed of 85/15 ...poly(lactide-co-glycolide) (PLAGA) have been evaluated for such uses. However, structural limitations may restrict the clinical use of these scaffolds. We have developed a novel sintered microsphere scaffold with a biomimetic pore system equivalent to the structure of trabecular bone. By modifying processing parameters, several different sintered microsphere structures were fabricated. Optimization of the structure dealt with modifications to sphere diameter and heating time
. Compressive testing illustrated a trend between microsphere diameter and modulus, where increased microsphere diameter resulted in decreased modulus. In addition, evaluation of the pore system showed a positive correlation between sphere diameter and pore diameter. Mercury porosimetry showed increased median pore size with an increased microsphere diameter. Heating time modifications showed that compressive modulus was dependent on the period of heating with longer heating times resulting in higher moduli. It was also shown that heating time did not affect the pore structure. Analysis of the structural data indicated that the microsphere matrix sintered for 4
h at a temperature of 160°C with a microsphere diameter of 600–710
μm resulted in an optimal, biomimetic structure with range in pore diameter of 83–300
μm, a median pore size of 210
μm, 35% porosity, and a compressive modulus of 232
MPa. An in vitro evaluation of human osteoblasts seeded onto the sintered matrix indicated that the structure was capable of supporting the attachment and proliferation of cells throughout its pore system. Immunofluorescent staining of actin showed that the cells were proliferating three-dimensionally through the pore system. The stain for osteocalcin was used and showed that cells maintained phenotypic expression for this bone specific protein. Through this work, it was shown that an osteoconductive PLAGA scaffold with a pore system used as a reverse template to the structure of trabecular bone could be fabricated through the sintered microsphere method.
In the development of 3-dimensional cell-polymer matrices for tissue engineering, the ability of osteoblast cells to maintain their phenotypic properties and form a mineralized matrix while seeded on ...the polymer surface is very important. Osteoblast cell differentiation and bone formation using rat calvaria cells were studied on the surface of a porous poly(lactide/glycolide)/hydroxyapatite (PLAGA/HA) 3-dimensional polymer matrix. Cell adhesion and proliferation were determined at 24 hr, 3, 7, 14, and 21 days. Cell attachment and proliferation were observed to increase throughout the first two weeks of the study, followed by a period of gradual plateauing of cell numbers. Environmental scanning electron microscopy demonstrated that cells grown on the surface of the 3-dimensional porous PLAGA/HA matrix retained their characteristic morphology and grew in a multi-layer fashion. Light microscopy observations of experiment cultures revealed active osteoblastic cells forming a characteristic mineralized matrix in the presence of β-glycerophosphate as a phosphate donor. Mineralization did not occurred in media either not supplemented with β-glycerophosphate or when the matrix without cells was incubated with the reagents, indicating that the mineralization was due to the cells and not the HA in the matrix. These results suggest that the 3-dimensional PLAGA/HA matrix could provide a matrix for bone cell differentiation and mineralization
in vitro and, therefore, may be a candidate as a synthetic implant for bone regeneration.
Recently, several studies have suggested the radiosensitizing effect of taxol, a microtubular inhibitor. Our overall hypothesis is that a combination of radiation and taxol may demonstrate ...therapeutic efficacy over doses of either individually. Studies examining taxol use have mostly focused on systemic administration, which can lead to undesired effects. To circumvent these side effects, we propose a locally administered polymeric microsphere delivery system combined with radiation therapy for the treatment of Ewing’s sarcoma. The present study focuses on the in vitro ability of taxol when present as a microencapsulated drug delivery system, and delivered locally at the site of the sarcoma/tumor, to block cells in the G2/M phase of the cell cycle and potentially enhance the radiation sensitivity of cells. Using the bioresorbable poly(anhydride-
co-imide), polypyromellityl-imidoalanine-1,6-bis(carboxy-phenoxy)hexane (PMA-CPH), and the radiosensitizing agent taxol, a microsphere based delivery system was fabricated. A solvent evaporation technique was used to encapsulate taxol at doses of 1%, 5%, and 10% in PMA-CPH microspheres. Release kinetics studies demonstrated that the total amount of taxol released and the release rate were directly dependent on loading percentage. Taxol’s bioactivity and radiosensitizing ability were measured using flow cytometry. Co-culture of Ewing’s sarcoma cells with and without taxol-loaded microspheres demonstrated that released taxol retained its bioactivity and effectively blocked cells in the radiosensitive G2/M phase of mitosis. The taxol-radiation delivery system studied achieved an 83% decrease in tumor cell count compared to control. Taxol effectively sensitized Ewing’s sarcoma cells to radiation with radiosensitivity shown to be independent of radiation dose at levels of dosages studied. This work has demonstrated that taxol can be effectively released from a biodegradable PMA-CPH microsphere delivery system while maintaining potent combined cytotoxic and radiosensitizing abilities.
In vitro assessment of the effects of platelet-rich plasma on the extracellular matrix metabolism of porcine intervertebral disc cells.
To determine whether platelet-rich plasma is effective in ...stimulating cell proliferation and extracellular matrix metabolism by porcine disc cells cultured in alginate beads.
Platelet-rich plasma is used to accelerate wound healing and tissue regeneration. Activated platelets release multiple growth factors that regulate cell proliferation, differentiation, and morphogenesis. Individual growth factors present in platelet-rich plasma have been demonstrated to affect the metabolism of intervertebral disc cells.
Platelet-poor and platelet-rich plasma was isolated from fresh porcine blood using a commercially available platelet concentration system. After preculture for 7 days and serum starvation for 24 hours, the beads containing nucleus pulposus and anulus fibrosus cells were then cultured for another 72 hours in serum-free medium, 10% fetal bovine serum, 10% platelet-poor plasma, or 10% platelet-rich plasma. The synthesis of proteoglycans and collagen, the accumulation of proteoglycans, and the DNA content were biochemically assessed.
Platelet-rich plasma had a mild stimulatory effect on cell proliferation of intervertebral disc cells. Platelet-rich plasma treatment significantly upregulated proteoglycan and collagen synthesis and proteoglycan accumulation when compared with platelet-poor plasma.
Platelet-rich plasma was effective in stimulating cell proliferation and extracellular matrix metabolism. The response to platelet-rich plasma was greater in the case of anulus fibrosus cells than of nucleuspulposus cells. The local administration of platelet-rich plasma might stimulate intervertebral disc repair. In addition, given the risks of using animal serum for tissue engineering, autologous blood may gain favor as a source of growth factors and serum supplements needed for stimulating cells to engineer intervertebral disc tissues.
The need for synthetic alternatives to conventional bone grafts is due to the limitations of current grafting materials. Our approach has been to design polymer-based graft substitutes using ...microsphere technology. The gel microsphere matrix and the sintered microsphere matrix were designed using the random packing of poly(lactide-
co-glycolide) microspheres to create a three-dimensional porous structure. The evaluation of these methods dealt with analysis of effects of matrix composition and processing. Matrices were evaluated structurally by scanning electron microscopy and porosimetry, and biomechanically by compression testing.
The evaluation revealed the high modulus of the gel microsphere matrix and the versatility of the sintered microsphere matrix. The gel microsphere matrix incorporated hydroxyapatite particles and had a Young's modulus of 1651
MPa, but structural analysis through SEM revealed a pore system less optimal for bone in-growth. The sintered microsphere matrices were fabricated without hydroxyapatite particles by thermally fusing polymeric microspheres into a three-dimensional array, possessing interconnectivity and a modulus range of 241 (±82)–349 (±89)
MPa. The sintered microsphere matrix demonstrated a connected pore system and mechanical properties in the mid-range of cancellous bone. Porosimetry data indicated that matrix pore diameter varied directly with microsphere diameter, while pore volume was independent of microsphere diameter in the range of diameters examined. The microsphere-based matrices show promise as polymeric substitutes for bone repair.
A degradable polymer-ceramic matrix for use as a bone graft material is described. The fabrication method used produces 3-dimensional macroporous matrices which are structurally similar to cancellous ...bone in their porosity, mechanically similar to cancellous bone in compressive elastic modulus and chemically comparable to the mineral matrix of bone in that they contain hydroxyapatite (HA). A 50:50 copolymer of poly(lactide/glycolide) (PLAGA) reinforced by a particulate calcium phosphate ceramic, HA, was used to create a matrix composed of polymeric microspheres. The channels between these spheres were pores approximately 100 microns in diameter. Four polymer/ceramic ratios were used in matrix fabrication: 1:0, 1:1, 2.5:1, and 5:1. The mechanical behavior of the material was found to vary with ceramic content. Increased levels of HA resulted in increased compressive elastic moduli. Prior to polymer degradation, moduli ranged from a high of 1459 MPa (50% HA) to a low of 293 MPa (0% HA). Degradation studies over a 6-week period showed that 0 and 16.7% HA-containing matrices lost up to 50% of their original weight, while the 28.6 and 50% IIA-containing matrices lost up to 20% of their original weight. Increased HA matrix content translated into decreased rates of matrix degradation. Environmental scanning electron microscopy (ESEM) confirmed that the polymer matrix contained pores that were interconnected during degradation. Viewed via ESEM, 10% HA containing matrices completely degraded by 6 weeks, while 50% HA matrices remained relatively stable. These studies indicate that the porous 3-dimensional polymer/ceramic matrix may potentially be useful as a synthetic material for bone repair.
Poly(anhydride-co-imides) are currently under study for applications involving bone. The cytotoxicity of a series of poly(anhydride-co-imides) with osteoblast-like cells (MC3T3-E1) was evaluated. The ...imide component of the copolymers was based on pyromellitylimidoalanine and the anhydride component was based on either sebacic acid or 1,6-bis(carboxyphenoxy)hexane. Cell adhesion and proliferation on the surfaces of the polymer discs were observed by environmental scanning electron microscopy. During the first 24 hours of attachment, the cells showed normal morphology when cultured on copolymers containing 1,6-bis(carboxyphenoxy)hexane. The cells did not adhere to the polymers containing sebacic acid, probably due to the rapid degradation of the polymer surfaces. Concurrently, the effects of polymer breakdown products on osteoblast-like cells were evaluated by studying their proliferation (cell numbers), viability (dye exclusion), morphology (light microscopy), and phenotypic expression. The morphology of osteoblast-like cells cultured in the presence of the polymer breakdown products pyromellitylimidoalanine and pyromellitic acid was found to be similar to that of the same cells grown on tissue culture polystyrene and consisted of a characteristic polygonal shape. With use of a monoclonal antibody to osteocalcin, these cells were shown to demonstrate preserved osteoblast phenotype with growth over a 21-day period. In addition, the cells reached confluency after 3-4 days, similar to cells grown on tissue culture polystyrene. This in vitro evaluation showed that the poly(anhydride-co-imides) evaluated are non-cytotoxic and may be viable biomaterials for orthopaedic applications.
The anterior cruciate ligament (ACL) is the most commonly injured intra-articular ligament of the knee, and limitations in existing reconstruction grafts have prompted an interest in tissue ...engineered solutions. Previously, we reported on a tissue-engineered ACL scaffold fabricated using a novel, three-dimensional braiding technology. A critical factor in determining cellular response to such a graft is material selection. The objective of this in vitro study was to optimize the braided scaffold, focusing on material composition and the identification of an appropriate polymer. The selection criteria are based on cellular response, construct degradation, and the associated mechanical properties. Three compositions of poly-
α-hydroxyester fibers, namely polyglycolic acid (PGA), poly-
L-lactic acid (PLLA), and polylactic-
co-glycolic acid 82:18 (PLAGA) were examined. The effects of polymer composition on scaffold mechanical properties and degradation were evaluated in physiologically relevant solutions. Prior to culturing with primary rabbit ACL cells, scaffolds were pre-coated with fibronectin (Fn, PGA-Fn, PLAGA-Fn, PLLA-Fn), an important protein which is upregulated during ligament healing. Cell attachment and growth were examined as a function of time and polymer composition. While PGA scaffolds measured the highest tensile strength followed by PLLA and PLAGA, its rapid degradation in vitro resulted in matrix disruption and cell death over time. PLLA-based scaffolds maintained their structural integrity and exhibited superior mechanical properties over time. The response of ACL cells was found to be dependent on polymer composition, with the highest cell number measured on PLLA-Fn scaffolds. Surface modification of polymer scaffolds with Fn improved cell attachment efficiency and effected the long-term matrix production by ACL cells on PLLA and PLAGA scaffolds. Therefore based on the overall cellular response and its temporal mechanical and degradation properties in vitro, the PLLA braided scaffold pre-coated with Fn was found to be the most suitable substrate for ACL tissue engineering.
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