Supplementation with platelet-rich plasma improves the in vitro formation of tissue-engineered cartilage with enhanced mechanical properties.

PURPOSE: This study aimed to determine the effects of platelet-rich plasma (PRP) on the histologic, biochemical, and biomechanical properties of tissue-engineered cartilage. METHODS: Chondrocytes isolated from bovine metacarpal-phalangeal articular cartilage were seeded on top of a porous ceramic substrate (calcium polyphosphate [CPP]). Cultures were supplemented with fetal bovine serum (FBS), PRP, or platelet-poor plasma (PPP) at 5%. On day 5, the concentration was increased to 20%. PRP and PPP were obtained through centrifugation of whole blood withdrawn from a mature cow. After 2 weeks, samples (n = 8) were analyzed histologically, biochemically, and biomechanically. Data were analyzed using the Wilcoxon test (significance, P < .05). RESULTS: Chondrocytes cultured in 20% PRP formed thicker cartilage tissue (1.6 ± 0.2 mm) than did cells grown in 20% FBS (0.7 ± 0.008 mm; P = .002) and 20% PPP (0.8 ± 0.2 mm; P = .03). Cartilage tissue generated in the presence of 20% PRP had a greater equilibrium modulus of 38.1 ± 3.6 kPa versus 15.6 ± 1.5 kPa (P = .0002) for 20% PPP and 20.4 ± 3.5 kPa (P = .007) for 20% FBS. Glycosaminoglycan (GAG) content was increased in tissues formed in 20% PRP (176 ± 18.8 µg GAG/mg) compared with those grown in 20% FBS (112 ± 10.6 µg GAG/mg; P = .01) or 20% PPP (131.5 ± 14.8 µg GAG/mg; P = .11). Hydroxyproline content was similar whether the media was supplemented with 20% PRP (8.7 ± 0.9 µg/mg), 20% FBS (7.6 ± 0.9 µg/mg; P = .37), or 20% PPP (6.4 ± 1 µg/mg; P = .28). DNA content was similar in all tissues whether formed in 20% PRP (11.9 ± 3.5 µg/mg), 20% FBS (9.3 ± 2.5 µg/mg; P = .99), or 20% PPP (7.2 ± 1.3 µg/mg; P = .78). Immunostained samples showed prevalence of type II collagen in tissues formed in the presence of 20% PRP. CONCLUSIONS: The presence of PRP in the culture media enhances the in vitro formation of cartilage, with increased GAG content and greater compressive mechanical properties, while maintaining characteristics of hyaline phenotype. CLINICAL RELEVANCE: Understanding the in vitro effects of PRP on tissue-engineered cartilage may lead to the creation of engineered cartilage tissue with enhanced properties suitable for cartilage repair.

Integration of tissue-engineered cartilage with host cartilage: an in vitro model.

BACKGROUND: We developed a tissue-engineered biphasic cartilage bone substitute construct which has been shown to integrate with host cartilage and differs from autologous osteochondral transfer in which integration with host cartilage does not occur. QUESTIONS/PURPOSES: (1) Develop a reproducible in vitro model to study the mechanisms regulating tissue-engineered cartilage integration with host cartilage, (2) compare the integrative properties of tissue-engineered cartilage with autologous cartilage and (3) determine if chondrocytes from the in-vitro formed cartilage migrate across the integration site. METHODS: A biphasic construct was placed into host bovine osteochondral explant and cultured for up to 8 weeks (n = 6 at each time point). Autologous osteochondral implants served as controls (n = 6 at each time point). Integration was evaluated histologically, ultrastructurally, biochemically and biomechanically. Chondrocytes used to form cartilage in vitro were labeled with carboxyfluorescein diacetate which allowed evaluation of cell migration into host cartilage. RESULTS: Histologic assessment demonstrated that tissue-engineered cartilage integrated over time, unlike autologous osteochondral implant controls. Biochemically there was an increase in collagen content of the tissue-engineered implant over time but was well below that for native cartilage. Integration strength increased between 4 and 8 weeks as determined by a pushout test. Fluorescent cells were detected in the host cartilage up to 1.5 mm from the interface demonstrating chondrocyte migration. CONCLUSIONS: Tissue-engineered cartilage demonstrated improved integration over time in contrast to autologous osteochondral implants. Integration extent and strength increased with culture duration. There was chondrocyte migration from tissue-engineered cartilage to host cartilage. CLINICAL RELEVANCE: This in vitro integration model will allow study of the mechanism(s) regulating cartilage integration. Understanding this process will facilitate enhancement of cartilage repair strategies for the treatment of chondral injuries.

Calcium regulates cyclic compression-induced early changes in chondrocytes during in vitro cartilage tissue formation.

A single application of cyclic compression (1kPa, 1Hz, 30min) to bioengineered cartilage results in improved tissue formation through sequential catabolic and anabolic changes mediated via cell shape changes that are regulated by α5ß1 integrin and membrane-type metalloprotease (MT1-MMP). To determine if calcium was involved in this process, the role of calcium in regulating cell shape changes, MT1-MMP expression and integrin activity in response to mechanical stimulation was examined. Stimulation-induced changes in cell shape and MT1-MMP expression were abolished by chelation of extracellular calcium, and this effect was reversed by re-introduction of calcium. Spreading was inhibited by blocking stretch-activated channels (with gadolinium), while retraction was prevented by blocking the L-Type voltage-gated channel (with nifedipine); both compounds inhibited MT1-MMP upregulation. Calcium A23187 ionophore restored cellular response further supporting a role for these channels. Calcium regulated the integrin-mediated signalling pathway, which was facilitated through Src kinase. Both calcium- and integrin-mediated pathways converged on ERK-MAPK in response to stimulation. While both integrins and calcium signalling mediate chondrocyte mechanotransduction, calcium appears to play the major regulatory role. Understanding the underlying molecular mechanisms involved in chondrocyte mechanotransduction may lead to the development of improved bioengineered cartilage.

Solid freeform fabrication and characterization of porous calcium polyphosphate structures for tissue engineering purposes.

Solid freeform fabrication (SFF) enables the fabrication of anatomically shaped porous components required for formation of tissue engineered implants. This article reports on the characterization of a three-dimensional-printing method, as a powder-based SFF technique, to create reproducible porous structures composed of calcium polyphosphate (CPP). CPP powder of 75-150 microm was mixed with 10 wt % polyvinyl alcohol (PVA) polymeric binder, and used in the SFF machine with appropriate settings for powder mesh size. The PVA binder was eliminated during the annealing procedure used to sinter the CPP particles. The porous SFF fabricated components were characterized using scanning electron microscopy, micro-CT scanning, X-ray diffraction, and mercury intrusion porosimetry. In addition, mechanical testing was conducted to determine the compressive strength of the CPP cylinders. The 35 vol % porous structures displayed compressive strength on average of 33.86 MPa, a value 57% higher than CPP of equivalent volume percent porosity made through conventional gravity sintering. Dimensional deviation and shrinkage analysis was conducted to identify anisotropic factors required for dimensional compensation during SFF sample formation and subsequent sintering. Cell culture studies showed that the substrate supported cartilage formation in vitro, which was integrated with the top surface of the porous CPP similar to that observed when chondrocytes were grown on CPP formed by conventional gravity sintering methods as determined histologically and biochemically.

Articular cartilage subpopulations respond differently to cyclic compression in vitro.

The inferior biomechanical properties of in vitro-formed tissue remain a significant obstacle in bioengineering articular cartilage tissue. We have previously shown that cyclic compression (30 minutes, 1 kPa, 1 Hz) of chondrocytes isolated from full-thickness cartilage can induce greater matrix synthesis, although articular cartilage is composed of different subpopulations of chondrocytes, and their individual contribution to enhanced tissue formation has not been fully characterized. This study examines the contribution of chondrocyte subpopulations to this response. Bovine articular chondrocytes were isolated from superficial to mid zones (SMZs) or deep zones (DZs), placed in three-dimensional culture, and subjected to cyclic compression. DZ chondrocytes on calcium polyphosphate substrates formed thicker tissue than those from SMZs. Compression increased matrix accumulation in SMZ chondrocytes while decreasing accumulation in DZ chondrocytes. The SMZ and DZ chondrocytes also differed in their type 1 membrane-bound matrix metalloproteinase (MMP) and MMP-13 expression, enzymes that play a crucial role in mediating the response to mechanical stimulation. In addition, the duration of the culture period was important in determining the DZ response, raising the possibility that matrix accumulation plays a role in the response to stimulation. Understanding the cellular response to mechanical stimulation during tissue formation will facilitate our understanding of tissue growth and allow for further optimization of cartilage tissue formation in vitro.

Cold acclimation-induced up-regulation of the ribosomal protein L7 gene in the freeze tolerant wood frog, Rana sylvatica.

Natural freezing survival by the wood frog, Rana sylvatica, involves multiple organ-specific changes in gene expression. The present study used differential display PCR to find cold-responsive genes in wood frog skin. A cDNA was retrieved from skin that was in higher amounts in cold- versus warm-acclimated frogs. The cDNA was used to probe a wood frog liver cDNA library and retrieve a long sequence that, after the further application of 5'RACE, was shown to encode the full sequence of the ribosomal large subunit protein 7 (RPL7) (GenBank accession number AF175983). Wood frog RPL7 contained 246 amino acids and shared 90% identity with Xenopus laevis RPL7, 82-83% with chicken and zebrafish homologues, and 79% with mammalian RPL7. Multiple binding domains found in human RPL7 showed differing degrees of conservation in the frog protein. Transcript levels of rpl7 were elevated up to 4-fold in skin of cold-acclimated frogs as compared with warm-acclimated animals. Organ-specific responses by rpl7 transcripts also occurred when frogs were given survivable freezing exposures. Transcripts rose by 1.8-3.3 fold in brain and skeletal muscle during freezing but were unaffected in central organs such as liver and heart. Up-regulation of rpl7 also occurred in brain of anoxia-exposed frogs and RPL7 protein levels increased strongly in heart under both freezing and dehydration stresses. Cold- and freezing-responsive up-regulation of the rpl7 gene and RPL7 protein in selected organs suggests that targeted changes in selected ribosomal proteins may be an integral part of natural freeze tolerance.

Upregulation of the mitochondrial phosphate carrier during freezing in the wood frog Rana sylvatica: potential roles of transporters in freeze tolerance.

Screening of a cDNA library prepared from liver of the freeze-tolerant wood frog (Rana sylvatica) identified a freeze-responsive clone containing a 1370-nt sequence with an open reading frame of 360 amino acids. Sequence analysis revealed 84-86% identity with the mammalian inorganic phosphate carrier (PiC) that spans the inner mitochondrial membrane. Northern blot analysis showed that pic transcript levels increased over a time course of freezing, reaching 60-fold upregulation after 24-h frozen. Transcript levels were also assessed under freezing-related stresses with results showing a strong increase in pic transcript levels in response to dehydration (elevated 9.0-fold in 40% dehydrated frogs) but not under anoxia. Western blotting revealed elevated PiC protein over a time course of freeze-thaw whereas other mitochondrial carriers (dicarboxylate carrier, oxoglutarate transporter) of the same family were not affected by freezing. This modulation of PiC protein levels may play a role in mitochondrial ionic and/or osmotic balance during freeze-induced cell volume reduction.