Thomas M. Hering

BMP‐2 induction and TGF‐β1 modulation of rat periosteal cell chondrogenesis

Periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondrocytes during normal bone growth and fracture healing. TGF‐β1 and BMP‐2 have been implicated in the regulation of the chondrogenic differentiation of these cells, but their roles are not fully defined. This study was undertaken to investigate the chondrogenic effects of TGF‐β1 and BMP‐2 on rat periosteum‐derived cells during in vitro chondrogenesis in a three‐dimensional aggregate culture. RT‐PCR analyses for gene expression of cartilage‐specific matrix proteins revealed that treatment with BMP‐2 alone and combined treatment with TGF‐β1 and BMP‐2 induced time‐dependent mRNA expression of aggrecan core protein and type II collagen. At later times in culture, the aggregates treated with BMP‐2 exhibited expression of type X collagen and osteocalcin mRNA, which are markers of chondrocyte hypertrophy. Aggregates incubated with both TGF‐β1 and BMP‐2 showed no such expression. Treatment with TGF‐β1 alone did not lead to the expression of type II or X collagen mRNA, indicating that this factor itself did not independently induce chondrogenesis in rat periosteal cells. These data were consistent with histological and immunohistochemical results. After 14 days in culture, BMP‐2‐treated aggregates consisted of many hypertrophic chondrocytes within a metachromatic matrix, which was immunoreactive with anti‐type II and type X collagen antibodies. In contrast, at 14 days, TGF‐β1+BMP‐2‐treated aggregates did not contain any morphologically identifiable hypertrophic chondrocytes and their abundant extracellular matrix was not immunoreactive to the anti‐type X collagen antibody. Expression of BMPR‐IA, TGF‐β RI, and TGF‐β RII receptors was detected at all times in each culture condition, indicating that the distinct responses of aggregates to BMP‐2, TGF‐β1 and TGF‐β1+BMP‐2 were not due to overt differences in receptor expression. Collectively, our results suggest that BMP‐2 induces neochondrogenesis of rat periosteum‐derived cells, and that TGF‐β1 modulates the terminal differentiation in BMP‐2 induced chondrogenesis. J. Cell. Biochem. 80:284–294, 2001.

Type V collagen during granulation tissue development

The collagen content, as determined by hydroxyproline assay, of experimental granulation tissue in rats was observed to increase rapidly 21 days, and less rapidly to 90 days of tissue development. Resistance of the collagen to pepsin digestion reached a maximum at 21 days, suggesting more extensive or more stable crosslinking at that time. Type V collagen and the expected collagen types I and III were present in pepsin extracts of the granulation tissue as determined by SDS-polyacrylamide gel electrophoresis. Over 3 months of tissue development the relative quantity of type V collagen, as evidenced by changes in the alpha B chain, varied in parallel with the changing vascularity of the tissue, suggesting an association with capillary endothelial cells and angiogenesis.

Changes of chondrocyte expression profiles in human MSC aggregates in the presence of PEG microspheres and TGF-β3

Biomaterial microparticles are commonly utilized as growth factor delivery vehicles to induce chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs). To address whether the presence of microparticles could themselves affect differentiation of MSCs, a 3D co-aggregate system was developed containing an equal volume of human primary bone marrow-derived MSCs and non-degradable RGD-conjugated poly(ethylene glycol) microspheres (PEG-μs). Following TGF-β3 induction, differences in cell phenotype, gene expression and protein localization patterns were found when compared to MSC aggregate cultures devoid of PEG-μs. An outer fibrous layer always found in differentiated MSC aggregate cultures was not formed in the presence of PEG-μs. Type II collagen protein was synthesized by cells in both culture systems, although increased levels of the long (embryonic) procollagen isoforms were found in MSC/PEG-μs aggregates. Ubiquitous deposition of type I and type X collagen proteins was found in MSC/PEG-μs cultures while the expression patterns of these collagens was restricted to specific areas in MSC aggregates. These findings show that MSCs respond differently to TGF-β3 when in a PEG-μs environment due to effects of cell dilution, altered growth factor diffusion and/or cellular interactions with the microspheres. Although not all of the expression patterns pointed toward improved chondrogenic differentiation in the MSC/PEG-μs cultures, the surprisingly large impact of the microparticles themselves should be considered when designing drug delivery/scaffold strategies.

Phenotypic modulation of newly synthesized proteoglycans in human cartilage and chondrocytes

The proteoglycans synthesized by human osteoarthritic femoral head cartilage and nonarthritic articular cartilage age-matched to the osteoarthritic cartilage specimens was studied in explant cultures and in chondrocytes generated by explant outgrowth from the cartilages. Twenty-four hours after explanation, both nonarthritic articular cartilage and osteoarthritic cartilage synthesized principally one large proteoglycan core protein that migrated on 3-5% acrylamide gels with an apparent molecular mass (M(r)) of approximately 520 kDa after enzymatic digestion with chondroitinase ABC and keratanase. The proteoglycan was found in both the explant itself and in the medium compartment of the culture as well. This proteoglycan contained chondroitin-6-sulfate, keratan sulfate and the hyaluronan binding region as evidenced by immunoblotting with murine anti-proteoglycan monoclonal antibodies indicating that the proteoglycan was aggrecan. To a much lesser extent two additional proteoglycan core proteins were also found in the explant but were not seen in the culture medium compartment. These proteoglycans possessed apparent M(r)s of approximately 480 kDa and approximately 390 kDa on 3-5% acrylamide gels after chondroitinase ABC and keratanase digestion. The medium compartment contained principally the approximately 520 kDa proteoglycan core protein. In osteoarthritic cartilage explants, the pattern of newly synthesized proteoglycans recovered from the tissue as assessed on 3-16% polyacrylamide gradient gels remained relatively the same from day 1 after explantation up to 36 days of culture. By contrast, the proteoglycans recovered from the culture medium contained chondroitin sulfate and keratan sulfate after 1, 7, and 21 days in culture but by 36 days appeared to contain only chondroitin sulfate. Chondrocytes generated from osteoarthritic cartilage and age-matched nonarthritic articular cartilage synthesized different patterns of large (greater than 200 kDa) proteoglycan. Whereas chondrocytes derived from osteoarthritic cartilage continued to synthesize principally the approximately 520 kDa proteoglycan core protein, the chondrocytes derived from nonarthritic cartilage synthesized in addition to this proteoglycan, abundant amounts of the other two proteoglycan core proteins as well.

Comparison of sensory neuron growth cone and filopodial responses to structurally diverse aggrecan variants, in vitro.

Following spinal cord injury, a regenerating neurite encounters a glial scar enriched in chondroitin sulfate proteoglycans (CSPGs), which presents a major barrier. There are two points at which a neurite makes contact with glial scar CSPGs: initially, filopodia surrounding the growth cone extend and make contact with CSPGs, then the peripheral domain of the entire growth cone makes CSPG contact. Aggrecan is a CSPG commonly used to model the effect CSPGs have on elongating or regenerating neurites. In this study, we investigated filopodia and growth cone responses to contact with structurally diverse aggrecan variants using the common stripe assay. Using time-lapse imaging with 15-s intervals, we measured growth cone area, growth cone width, growth cone length, filopodia number, total filopodia length, and the length of the longest filopodia following contact with aggrecan. Responses were measured after both filopodia and growth cone contact with five different preparations of aggrecan: two forms of aggrecan derived from bovine articular cartilage (purified and prepared using different techniques), recombinant aggrecan lacking chondroitin sulfate side chains (produced in CHO-745 cells) and two additional recombinant aggrecan preparations with varying lengths of chondroitin sulfate side chains (produced in CHO-K1 and COS-7 cells). Responses in filopodia and growth cone behavior differed between the structurally diverse aggrecan variants. Mutant CHO-745 aggrecan (lacking chondroitin sulfate chains) permitted extensive growth across the PG stripe. Filopodia contact with the CHO-745 aggrecan caused a significant increase in growth cone width and filopodia length (112.7% ± 4.9 and 150.9% ± 7.2 respectively, p<0.05), and subsequently upon growth cone contact, growth cone width remained elevated along with a reduction in filopodia number (121.9% ± 4.2; 72.39% ± 6.4, p<0.05). COS-7 derived aggrecan inhibited neurite outgrowth following growth cone contact. Filopodia contact produced an increase in growth cone area and width (126.5% ± 8.1; 150.3% ± 13.31, p<0.001), and while these parameters returned to baseline upon growth cone contact, a reduction in filopodia number and length was observed (73.94% ± 5.8, 75.3% ± 6.2, p<0.05). CHO-K1 derived aggrecan inhibited neurite outgrowth following filopodia contact, and caused an increase in growth cone area and length (157.6% ± 6.2; 117.0% ± 2.8, p<0.001). Interestingly, the two bovine articular cartilage aggrecan preparations differed in their effects on neurite outgrowth. The proprietary aggrecan (BA I, Sigma-Aldrich) inhibited neurites at the point of growth cone contact, while our chemically purified aggrecan (BA II) inhibited neurite outgrowth at the point of filopodia contact. BA I caused a reduction in growth cone width following filopodia contact (91.7% ± 2.5, p<0.05). Upon growth cone contact, there was a further reduction in growth cone width and area (66.4% ± 2.2; 75.6% ± 2.9; p<0.05), as well as reductions in filopodia number, total length, and max length (75.9% ± 5.7, p<0.05; 68.8% ± 6.0; 69.6% ± 3.5, p<0.001). Upon filopodia contact, BA II caused a significant increase in growth cone area, and reductions in filopodia number and total filopodia length (115.9% ± 5.4, p<0.05; 72.5% ± 2.7; 77.7% ± 3.2, p<0.001). In addition, filopodia contact with BA I caused a significant reduction in growth cone velocity (38.6 nm/s ± 1.3 before contact, 17.1 nm/s ± 3.6 after contact). These data showed that neuron morphology and behavior are differentially dependent upon aggrecan structure. Furthermore, the behavioral changes associated with the approaching growth cone may be predictive of inhibition or growth.

Collagen Type Distribution in Healing of Synthetic Arterial Prostheses

Layers of tissue encapsulating vascular prostheses recovered from humans were extracted and analyzed by SDS-polyacrylamide gel electrophoresis to determine the distribution of genetically distinct collagen types. Type V collagen was in maximal concentration in extracts of tissues nearest to the prosthesis lumen, type III in extracts of chronically inflamed tissue filling the interstices of the porous prosthesis, and type I in extracts of fibrous occlusive or outer capsule tissue. This pattern of distribution of collagen types across the prosthesis wall may have arisen due to the influence of modulating factors originating in the blood flowing through the prosthesis, and factors produced by inflammatory cells chronically present at the tissue-biomaterial interface. The increased proportion of type V collagen at or near the lumen may contribute to the recognized antithrombogenic properties of human pseudointima.

THE HEALING RESPONSE AND VASCULAR GRAFTS

Publisher Summary This chapter discusses the healing response and vascular grafts. The blood-materials interaction in human vascular grafts is desirable as this information provide the basis for the development of better porous grafts. Blood will not result in thrombus formation or cause biochemical or physiological damage to the cells or proteins. The blood-materials interaction for vascular graft materials and involves the healing phenomenon in the vascular grafts. The retrieval of human vascular grafts offers the opportunity to study the various interrelationships in the healing response to vascular grafts. Various studies discussed in the chapter are a part of a larger program directed toward determining the composition of the pseudointima that develops in human vascular grafts and in vascular grafts placed in other species, determining specific alterations or variations in the patients blood protein and cellular levels immediately postimplantation of a vascular graft, determining the magnitude of platelet activation in an in vitro pump system which will provide in vitro–in vivo correlations and developing the information base of the Cleveland Vascular Society.