Kawamura M, Urist M R
Department of Surgery, University of California, Los Angeles 90024.
Dev Biol. 1988 Dec;130(2):435-42. doi: 10.1016/0012-1606(88)90339-9.
Connective tissue outgrowths of neonatal muscle onto a substratum of bone matrix differentiate into cartilage in response to a bone morphogenetic protein (BMP). The BMP can be separated from bone matrix by extraction with 4 M guanidine hydrochloride (GuHCl) or degraded in situ by endogenous proteolytic enzymes to deactivate the matrix. Rat triceps muscle was minced in a suspension of noncollagenous bone matrix proteins including BMP (BMP/NCP) in culture medium. To investigate the possible synergistic interactions in induced chondrogenesis, six biosynthesized, highly purified growth factors were similarly added to the culture alone or in combination with BMP. Human interleukin-1 (IL-1) and Forskolin were also introduced to test the effects on BMP/NCP-induced chondrogenesis. On Day 14 of cultivation, [3H]thymidine incorporation into DNA and [35S]sulfate incorporation into glycosaminoglycans (GAG) were measured, and the values were expressed as percentages of the control. The quantity of induced cartilage formation was estimated by a histomorphometric scoring system. Under the influence of BMP/NCP, cultures grew on deactivated matrix, incorporated 55% more [3H]thymidine into DNA, incorporated 115% more [35S]sulfate into GAG than control cultures, and differentiated into cartilage. Without BMP/NCP, growth factors, IL-1, and Forskolin did not produce a comparable incorporation of either [3H]thymidine or [35S]sulfate, and they induced differentiation of fibrous tissue only. In the presence of BMP/NCP, cartilage developed in nearly all cultures. When the media were supplemented with growth factors, measurable increases in uptake of [3H]thymidine occurred with human epidermal growth factor (h-EGF), insulin-like growth factor-1 (IGF-1), nerve growth factor (NGF), transforming growth factor-beta (TGF-beta), bovine acidic fibroblast growth factor (baFGF), IL-1, bovine basic fibroblast growth factor (bbFGF), and Forskolin. Measurable increases in uptake of [35S]sulfate into GAG occurred with IL-1, baFGF, TGF-beta, h-EGF, IGF-1, bbFGF, NGF, and Forskolin. Synergistic interaction with BMP was considered when the quantity of cartilage developed (on a scale of 0-12 scores) in excess of the quantity of Score 4 induced by BMP/NCP alone. A cytokine, IL-1, had the greatest effect (Score 9). TGF-beta (Score 7), baFGF (Score 6), and NGF (Score 6) had relatively little effect. h-EGF, IGF-1, bbFGF, and Forskolin had no effect on cartilage development.(ABSTRACT TRUNCATED AT 400 WORDS)
新生肌肉在骨基质底物上的结缔组织生长物会因骨形态发生蛋白（BMP）而分化为软骨。BMP可通过用4M盐酸胍（GuHCl）提取从骨基质中分离出来，或被内源性蛋白水解酶原位降解以使基质失活。将大鼠肱三头肌在含有包括BMP在内的非胶原蛋白骨基质蛋白（BMP/NCP）的培养基悬浮液中切碎。为了研究诱导软骨形成过程中可能的协同相互作用，将六种生物合成的、高度纯化的生长因子单独或与BMP联合添加到培养物中。还引入了人白细胞介素-1（IL-1）和福斯可林以测试对BMP/NCP诱导的软骨形成的影响。在培养的第14天，测量[3H]胸腺嘧啶核苷掺入DNA和[35S]硫酸盐掺入糖胺聚糖（GAG）的情况，并将这些值表示为对照的百分比。通过组织形态计量评分系统估计诱导的软骨形成量。在BMP/NCP的影响下，培养物在失活的基质上生长，与对照培养物相比，DNA中[3H]胸腺嘧啶核苷掺入量多55%，GAG中[35S]硫酸盐掺入量多115%，并分化为软骨。没有BMP/NCP时，生长因子、IL-1和福斯可林不会产生类似的[3H]胸腺嘧啶核苷或[35S]硫酸盐掺入，它们仅诱导纤维组织分化。在BMP/NCP存在的情况下，几乎所有培养物中都形成了软骨。当培养基中添加生长因子时，人表皮生长因子（h-EGF）、胰岛素样生长因子-1（IGF-1）、神经生长因子（NGF）、转化生长因子-β（TGF-β）、牛酸性成纤维细胞生长因子（baFGF）、IL-1、牛碱性成纤维细胞生长因子（bbFGF）和福斯可林使[3H]胸腺嘧啶核苷摄取量有可测量的增加。IL-1、baFGF、TGF-β、h-EGF、IGF-1、bbFGF、NGF和福斯可林使GAG中[35S]硫酸盐摄取量有可测量的增加。当形成的软骨量（以0至12分评分）超过单独由BMP/NCP诱导的4分的量时，考虑与BMP的协同相互作用。一种细胞因子IL-1的作用最大（9分）。TGF-β（7分）、baFGF（6分）和NGF（6分）的作用相对较小。h-EGF、IGF-1、bbFGF和福斯可林对软骨发育没有影响。（摘要截断于400字）
