Epidermal lipid metabolism of cultured skin substitutes during healing of full-thickness wounds in athymic mice

Cultured epidermal keratinocytes provide an abundant supply of biologic material for wound treatment. Because restoration of barrier function is a definitive criterion for efficacy of wound closure and depends on the lipids present in the epidermis, we analyzed lipid composition of the epidermis in cultured skin substitutes in vitro and after grafting to athymic mice. The cultured skin substitutes were prepared from human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates. After 14 days of incubation, cultured skin substitutes were grafted orthotopically onto full-thickness wounds in athymic mice. Samples for lipid analysis were collected after 14 and 34 days of in vitro incubation, and 3 weeks and 4 months after grafting. Both in vitro samples show disproportions in epidermal lipid profile as compared with the native human epidermis, i.e., a low amount of phospholipids (indicating imbalance in proliferation and differentiation); a large excess of triglycerides (storage lipids); and low levels of free fatty acids, gluco-sphingolipids, cholesterol sulfate, and ceramides-suggesting abnormal composition of stratum corneum barrier lipids. Fatty acid analysis of cultured skin substitutes in vitro revealed insufficient uptake of linoleic acid, which resulted in increased synthesis of and substitution with monounsaturated fatty acids, mainly oleic acid. These abnormalities were partially corrected by 3 weeks after grafting; and 4 months after grafting, all epidermal lipids, with some minor exceptions, were synthesized in proportions very similar to human epidermis. Results of this study show that grafting of cultured skin substitutes to a physiologic host permits the recovery of lipid in proportion to that required for barrier formation in normal human epidermis.     

Studies in fetal wound healing: II. A fetal environment accelerates fibroblast migration in vitro

We have used an in vitro model of wound healing using scratches made in a confluent monolayer of fibroblasts. The effects of fetal calf and postnatal calf serum on the migration of fibroblasts were compared. Differences between fetal and calf serum-incubated fibroblasts grown on coverslips were observed within 15 minutes of exposure. Cells in fetal serum began to change both shape and orientation and to move into the trough created by the scratch. The fibroblasts incubated in fetal calf serum completely filled in the trough within 16 hours while those incubated in calf serum did not do so even after 24 hours. We estimate that, at any point, there was a 50% lag time in the migration of the fibroblasts in the presence of postnatal calf serum. This difference in migration and filling was not a function of mitogenesis; the mitogenicity of the two sera were comparable. The results suggest that fibroblast migration in vitro is accelerated by the fetal serum. A similar mechanism may occur in vivo and may underlie the ability of the fetal wound to heal more rapidly.     

Tissue culture of human fetal pancreas. Effects of human serum on development and endocrine function of isletlike cell clusters

The human fetal pancreas represents a source of insulin-producing beta-cells with a potential for transplantation to diabetic patients. It has previously been shown that such cells can be viably maintained in tissue culture media containing fetal calf serum (FCS) and that these explants continue to synthesize and release insulin. In this study the effects of human serum (HS) on the growth and function of human fetal pancreatic explants have been compared with those of FCS. For this purpose, pancreatic glands, obtained after prostaglandin-induced abortions, were briefly exposed to collagenase, and the digest was cultured in RPMI-1640 medium plus 10% pooled HS or FCS. The outgrowth of isletlike cell clusters (ICCs) was monitored. In 31 of 58 consecutively explanted glands, development of ICCs was observed. In the presence of FCS the outgrowth of ICC took place on top of a fibroblast monocellular cell layer; HS effected less growth of fibroblasts and increased the formation of ICCs about sevenfold compared with explants from the same glands maintained in FCS. However, in the explant cultures with HS, the cell number per ICC, expressed as DNA content, was reduced by 50%. In both FCS and HS the insulin content of the medium showed great variability and progressively declined from day 2 to day 5. The medium glucagon concentration also decreased but not to the same extent as that of insulin. Immunocytochemical-stained ICCs showed insulin- and glucagon-positive cells scattered among most nonstained, presumably nonendocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Up-regulation by human recombinant transforming growth factor beta-1 of collagen production in cultured dermal fibroblasts is mediated by the inhibition of nitric oxide signaling

BACKGROUND: Hypertrophic scarring remains the most disabling sequela for burn survivors. Little is known about its pathogenesis. Collagen accumulation, however, has been consistently observed in burn hypertrophic scars (HS). STUDY DESIGN: We have studied collagen production in the dermal fibroblasts derived from HS, which has developed for 9 months to 2 years. Reconstructive surgery was performed to remove HS from which the fibroblasts were cultured. Similarly, the normal cells were grown from the patient's donor site (DS), which provided autografting to the HS site. Collagen production in HS and DS fibroblasts was compared and analyzed in minimal essential amino acid medium containing 5% fetal bovine serum with inclusion of L-ascorbic acid (100 microg/mL) and beta-aminopropoinitrile (100 microg/mL) by monitoring a 20-h [3H]proline incorporation into bacterial collagenase III-digestible protein in the conditioned media. RESULTS: We failed to detect any significant difference in collagen production in vitro between HS and DS. Irrespective of the fibroblasts from HS or DS, collagen production was substantially stimulated by human recombinant transforming growth factor beta-1 (TGF-beta1) (20 ng/mL) by approximately 250% after a 3-day pretreatment. In contrast, sodium nitroprusside (SNP) at 100 microM exhibited significant suppression (68%), which was rescued by hemoglobin (10 microM). TGF-beta1 significantly decreased nitric oxide (NO) production by 55%. In contrast, NO level drastically increased by 350% following SNP treatment. Epidermal growth factor showed no effect on either collagen production or NO level. The linear regression analysis shows a significant inverse correlation (r = 0.72; p < 0.05) of NO level with collagen production, suggesting the involvement of NO signaling in the modulation of collagen production. Consistent with the notion, we further showed that N-nitro-L-arginine methyl ester (100 microM) caused a synergistic stimulation and an arrested inhibition of collagen production in the presence of TGFbeta-1 and SNP, respectively. 8-BrcGMP (300 microM) mimicked the NO inhibitory action, while methylene blue (50 microM) restored the collagen production which was inhibited by SNP. Moreover, 8-BrcGMP offset the stimulation of collagen production. CONCLUSIONS: The dermal fibroblasts derived from HS were not different from normals with respect to collagen production and their responses to regulations. The inhibition of collagen production was achieved by a cGMP-dependent NO action. TGFbeta-1 inhibited NO/cGMP signaling to ensure its stimulatory effect on collagen production in the dermal fibroblasts.     

Transient exposure to tumor necrosis factor-alpha inhibits collagen accumulation by cultured hypertrophic scar fibroblasts.

BACKGROUND: Hypertrophic scars (HS) are frequent consequences of deep dermal injury, such as deep partial-thickness burns and abrasions, and are characterized by overproduction of collagen. In vitro studies have shown that cultured HS fibroblasts produce elevated levels of collagen and insulin-like growth factor-binding protein 3 (IGFBP-3). Additionally, histological studies have indicated HS contain fewer tumor necrosis factor alpha (TNF-alpha)-positive infiltrating cells and express lower levels of TNF-alpha mRNA, suggesting TNF-alpha, which can inhibit collagen expression in some systems, may function to deactivate the wound healing process in scars. HS also exhibit increased levels of transforming growth factor beta (TGF-beta), a factor that stimulates collagen and extracellular matrix deposition by fibroblasts and also stimulates IGFBP-3 expression. In some systems, IGFBP-3 mediates the effects of TGF-beta. The present study sought to determine the effects of continuous and transient TNF-alpha exposure on collagen and IGFBP-3 expression by cultured HS fibroblasts and to investigate the role of IGFBP-3 in collagen accretion by HS fibroblasts. MATERIALS AND METHODS: Superficial and deep dermal HS fibroblasts from four patients were cultured. Fibroblasts were cultured in serum-free medium and exposed to 0-2 ng/ml TNF-alpha for 0, 1, 4, or 72 h. After 72 h of culture, medium samples were processed for Western blot analysis of type I collagen accumulation or for ligand blot analysis of IGFBP-3 accumulation. The effects of an anti-IGFBP-3 neutralizing antibody on collagen accumulation were also assessed. RESULTS: Treatment of superficial and deep HS fibroblasts with TNF-alpha resulted in dose-dependent decreases in accumulation of both type I collagen and IGFBP-3 in the culture medium (P < 0.01). However, using the anti-IGFBP-3 neutralizing antibody, a causal relationship between decreased IGFBP-3 and decreased collagen accumulation could not be demonstrated. Transient exposure of cultured HS fibroblasts to TNF-alpha for as little as 1 h was as effective as continuous exposure to TNF-alpha for 72 h in inhibiting collagen accumulation. CONCLUSIONS: These results support the hypothesis that TNF-alpha functions as a wound healing deactivation signal that is deficient in HS. Although TNF-alpha inhibited accretion of both collagen and IGFBP-3, the role of IGFBP-3 in HS remains unresolved. This study suggests that transient TNF-alpha exposure may be used to inhibit collagen overaccumulation in HS and that the timing of TNF-alpha exposure following dermal injury may not be critical for this inhibition.     

胎牛血清在体外培养肝细胞极性形成的作用

目的观察胎牛血清在体外培养肝细胞极性形成的作用。方法分别利用含胎牛血清和不含胎牛血清培养液培养成鼠肝细胞,并观察血清对肝细胞形态、特异性膜区域蛋白分布以及蛋白分泌的影响,以确定胎牛血清在体外肝细胞极性形成的作用。结果不含血清组,肝细胞形成肝板样结构,肝细胞膜区域蛋白特异性分布,肝细胞保持稳定的蛋白分泌并持续增长达2周之久。而含血清组肝细胞培养3d后,分化良好的小管样结构消失,肝细胞膜区域蛋白失去特异性分布,蛋白分泌逐渐减少。结论胎牛血清可阻止肝细胞极性的形成,对于肝细胞移植及生物人工肝支持具有重要意义。     

人睾丸组织体外睾酮分泌变化的研究

用组织块培养法对取自4例健康猝死青年男子的睾丸组织进行了14天的培养,并测定了睾酮水平。结果发现4例睾丸组织睾酮分泌量在第3～第4天降至最低点,此后逐浙恢复,在第9～10天恢复至第1天的水平,并维持至培养结束,其中2例睾酮分泌量明显升高。提示体外培养的睾丸组织有3～4天的适应期,其活力可维持14天。     

5,7,4′-三羟基异黄酮对增生性瘢痕成纤维细胞增殖细胞核抗原表达及细胞周期的影响

目的 观察5,7,4′-三羟基异黄酮(genistein)对增生性瘢痕成纤维细胞增殖细胞核抗原(proliferating cell nuclear antigen, PCNA)及细胞周期的影响,探讨5,7,4′-三羟基异黄酮抑制增生性瘢痕成纤维细胞增殖的机制.方法 分离培养人增生性瘢痕成纤维细胞,分别加入25、50、100 μmol/L浓度的5,7,4′-三羟基异黄酮共培养48 h,免疫细胞化学法观察成纤维细胞PCNA蛋白的表达,流式细胞术检测细胞周期的变化.结果 各组5,7,4′-三羟基异黄酮作用后细胞PCNA的表达均降低(P＜0.05),50 μmol/L及100 μmol/L浓度组的抑制作用最为显著(P＜0.01);随药物浓度的增加,G0～G1期细胞比例逐渐下降,G2～M期细胞比例增加,表明细胞分裂受到抑制;100 μmol/L组的S期细胞数量比例也有增加,并于G1期前出现亚二倍体凋亡峰.结论 5,7,4′-三羟基异黄酮可通过影响细胞分裂与DNA合成抑制瘢痕增生.     

Altered proliferative responses of dermal fibroblasts to TGF-beta1 may contribute to chronic venous stasis ulcer

PURPOSE: Venous ulcer fibroblasts demonstrate decreased proliferative responses to growth factor stimulation, suggesting cellular senescence. However, the role of chronic venous insufficiency (CVI) disease progression and extracellular matrix (ECM) proteins in agonist-induced cellular proliferation is ill-defined. We hypothesize that CVI-induced fibroblast proliferative resistance to growth factors worsens with disease progression and is regulated by the composition of ECM. METHODS: Fibroblast explants were isolated from biopsy specimens from two patients without CVI and 16 patients with CVI of the lower calf (LC) and lower thigh (LT) and stratified according to CEAP disease severity: non-CVI (NC; n = 2), class 2-3 (n = 5), class 4 (n = 5), class 5 (n = 3), and class 6 (n = 3). Proliferation experiments were standardized with a neonatal foreskin fibroblast cell line (HS68). A 10-day course and dose response experiment with 0, 0.5, 1.0, 2.5, 5, 10, and 20 ng/mL of transforming growth factor-beta(1) (TGF-beta(1)) demonstrated maximal cell proliferation at 5 ng/mL of TGF-beta(1) on day 4. Under these conditions, CVI dermal fibroblasts were challenged with and without TGF-beta(1) and evaluated for proliferative responses on plates coated with polystyrene, collagen, and fibronectin. RESULTS: No differences in unstimulated proliferation were observed in LT and LC fibroblasts from patients with class 2-3 disease and LT fibroblasts from patients with class 4 and 5 disease, compared with NC and HS68 cells. LC fibroblasts from patients with class 4 disease (P <.05) and class 5 disease (P <.001), and LC (P <.001), and LT fibroblasts from patients with class 6 disease (P <.001) proliferated to a lesser degree than did NC and HS68 cells. The diminished proliferation observed in class 4 LC cells was reversible with TGF-beta(1) stimulation (P <.004); however, class 5 and class 6 LC and LT fibroblasts did not respond to stimulation with TGF-beta(1). Collagen increased proliferation of HS68 cells with (P <.05) and without (P <.01) TGF-beta(1), compared with cells grown on polystyrene, but did not increase proliferative responses in NC or CVI fibroblasts with and without TGF-beta(1). Similarly, fibronectin increased proliferation of HS68 cells (P <.05) compared with cells grown on polystyrene, but did not alter proliferation in CVI fibroblasts. Fibronectin did seem to inhibit TGF-beta(1)-induced proliferation observed in class 4 LC cells. CONCLUSION: These data indicate that clinical disease progression correlates with cellular dysfunction. Fibroblasts from patients with class 2-3 disease retain their unstimulated and agonist- induced proliferative capacity, compared with NC and HS68 cells. The onset of inflammatory skin changes (class 4 and class 5 disease) diminishes agonist-induced proliferation, and ulcer formation (class 6 disease) severely inhibits it. In addition, the composition of ECM does not affect TGF-beta(1)-induced proliferation of fibroblasts in CVI.     

Identification of maturation-related wheat-germ lectin-binding proteins in the culture of human corpus epididymal epithelial cells

This study is designed to investigate the synthesis of maturation-related wheat germ agglutinin (WGA) binding glycoproteins in the human corpus epididymal epithelial cells by in vitro culture. Epithelial cells were isolated from the corpus of human epididymides and cultured with RPMI 1640 medium supplemented with 10% fetal calf serum in type IV collagen-coated dishes at 37 degrees C. The epithelial nature, presence of fibroblasts, WGA-binding sites, and existence of GP-83 were determined by an indirect immunocytochemical and histochemical staining technique. Proteins in the cultured cells were analyzed by SDS-PAGE and autoradiography. After culturing for 10 days, the cells were shown to be positive with epithelial cell-specific keratins but devoid of fibroblasts. WGA-binding granules and positive binding sites of GP-83 were also detected in the cytoplasm. Immunoblots of cell extracts probed with the anti-GP-83 antibody from seminal fluid revealed the sperm maturation-related glycoprotein GP-83. The results indicate that WGA-binding proteins may be synthesized by the corpus epididymal epithelial cells of human and GP-83 may play an important role in sperm maturation. This culture model may be suitable for the investigation on the biosynthesis and physiology of human epididymal principal cells in vitro.     

成体神经干细胞培养方法的建立

目的 探讨体外建立培养成体神经干细胞(MSC)的方法.方法 从6周龄大鼠脑组织中分离神经干细胞,用神经干细胞培养液[DMEM/F12培养液添加1%N2、2%B27、20 μg/L表皮生长因子(EGF)和20μg/L碱性成纤维细胞生长因子(bFGF)]使其稳定增殖,10%胎牛血清诱导其贴壁分化.倒置显微镜下观察神经干细胞形态学变化;流式细胞仪检测神经干细胞表面标记物巢蛋白(Nestin)、神经元特异性烯醇酶(NSE,成熟神经元的特异性标志)、半乳糖脑苷脂(Galc-C,成熟少突胶质细胞的标记物)的表达.结果 分离所得细胞能在体外传代培养,流式细胞仪检测显示Nestin阳性细胞为97.6%,细胞经胎牛血清诱导分化后能形成NSE、Galc-C阳性细胞.结论 采用无血清的神经干细胞培养液能培养出具有分化潜能的成体神经干细胞.     

Wound healing in a fetal,adult, and scar tissue model: A comparative study

Early gestation fetal wounds heal without scar formation. Understanding the mechanism of this scarless healing may lead to new therapeutic strategies for improving adult wound healing. The aims of this study were to develop a human fetal wound model in which fetal healing can be studied and to compare this model with a human adult and scar tissue model. A burn wound (10 × 2 mm) was made in human ex vivo fetal, adult, and scar tissue under controlled and standardized conditions. Subsequently, the skin samples were cultured for 7, 14, and 21 days. Cells in the skin samples maintained their viability during the 21‐day culture period. Already after 7 days, a significantly higher median percentage of wound closure was achieved in the fetal skin model vs. the adult and scar tissue model (74% vs. 28 and 29%, respectively, p<0.05). After 21 days of culture, only fetal wounds were completely reepithelialized. Fibroblasts migrated into the wounded dermis of all three wound models during culture, but more fibroblasts were present earlier in the wound area of the fetal skin model. The fast reepithelialization and prompt presence of many fibroblasts in the fetal model suggest that rapid healing might play a role in scarless healing.     

Interleukin‐33 encourages scar formation in murine fetal skin wounds

The magnitude of the inflammatory response after skin injury is important for determining whether wounds in developing fetal skin will heal scarlessly (minimal inflammation) or with prominent scars (robust inflammation). One class of inflammatory mediators gaining attention for their role in wound inflammation is alarmins. In the current study, the alarmin interleukin‐33 (IL‐33) was examined in a mouse model of fetal wound healing. IL‐33 expression was elevated in scar‐forming embryonic day 18 wounds compared to scarless embryonic day 15 wounds. Furthermore, injection of IL‐33 into embryonic day 15 wounds caused scarring when wounds were analyzed at 7 days postwounding. The introduction of IL‐33 into embryonic day 15 wounds did not induce statistically significant changes in the number of neutrophils, mast cells, or macrophages in vivo. However, IL‐33 treatment enhanced collagen expression in cultured fibroblasts derived from adult and fetal murine skin, suggesting that IL‐33 may directly stimulate fibroblasts. In vitro studies suggested that the stimulation of collagen production by IL‐33 in fibroblasts was partially dependent on NF‐κB activation. Overall, the data suggest an association between IL‐33 and scar formation in fetal wounds.     

丹参对增生性瘢痕成纤维细胞形态的影响及增殖抑制初探

目的探索丹参治疗增生性瘢痕的机理。方法将培养的增生性瘢痕成纤维细胞加入含丹参的培养液中培养２４ｈ,与未加药者对照比较。结果①含丹参组成纤维细胞由长梭形变为圆形,而培养上清中的乳酸脱氢酶（ＬＤＨ）活性及ＭＴＴ（四甲基偶氮唑）比色法结果,用药组与对照组均无显著差异（Ｐ＞０．０５）;②流式细胞仪检测各时相细胞ＤＮＡ水平：用药组Ｇ０＋Ｇ１期细胞的百分比明显高于对照组（Ｐ＜０．０１）,而Ｇ２＋Ｍ期百分比则显著低于对照组（Ｐ＜０．０１）。结论丹参能改变增生性瘢痕成纤维细胞形态,而不影响其活力并抑制其增殖。     

CHARACTERIZATION OF CULTURED BLADDER SMOOTH MUSCLE CELLS: ASSESSMENT OF IN VITRO CONTRACTILITY

PURPOSE: The contractile properties of in vitro cultured bladder smooth muscle cells (SMC) are unknown. This study characterized the in vitro contractile response of human and rat bladder SMC to several pharmacological agonists known to induce in vivo contraction of intact bladder muscle. MATERIALS AND METHODS: Human and rat bladder SMC were seeded separately within attached collagen lattices. Contractility of SMC was analyzed by measuring alterations in lattice diameter after exposure and release to the following contractile agonists: carbachol (10(-7)-10(-3) microM), calcium-ionophore (10 microM), lysophosphatidic acid (LPA) (1 microM), endothelin (0.1 microM), KCl (3.33 mmicroM) angiotensin II (10 microM), and serotonin (100 microM). Results were recorded as a mean reduction of the lattice diameter. In addition, immunohistochemical analysis for phenotypic markers of smooth muscle cell differentiation was performed on bladder SMC cultured within collagen lattices. Human palmar fascia fibroblasts, which have been previously well characterized by in vitro contractility and immunohistochemistry, were tested in parallel and used as controls for all the above experiments. RESULTS: Human SMC had significant contractile responses to calcium-ionophore (31% +/- 4 relative percent contraction, p <0.05), LPA (34% +/- 4, p <0.05), and endothelin (37 +/- 5%, p <05). There was no significant contraction in response to carbachol, angiotensin II, KCl, or serotonin. Rat bladder SMC had a similar contractile response but did not contract in response to endothelin. In contrast to human and rat bladder SMC, fibroblasts did not contract to calcium-ionophore. CONCLUSIONS: In vitro cultured bladder SMC demonstrate loss of contractile response to normal in vivo pharmacologic agonists. Both human and rat bladder SMC can be distinguished in vitro from fibroblasts based upon their lack of contractile response to calcium- ionophore. These results demonstrate the ability to further characterize cultured bladder SMC with in vitro contractility. Further characterization is essential if we are to advance our understanding of the clinical applicability of in vitro studies utilizing cultured bladder SMC.     

A study of bone marrow and subcutaneous fatty acid composition in subjects of varying bone mineral density

Osteoporosis is associated with an increase in marrow fat. Fats, particularly polyunsaturated fats, either in co-cultures or diet, have been shown to significantly influence bone remodeling. Whether the increase in marrow fat seen in osteoporosis is also associated with a change in fatty acid composition is not known. This study was undertaken to investigate the fatty acid composition in subjects of varying bone mineral density (BMD). Samples of marrow fat and subcutaneous fat from 126 subjects (98 females, 34 males, mean age 69.7 ± 10.5 years) undergoing orthopedic surgery were analyzed for fatty acid composition by gas chromatography. These results were correlated with BMD assessed by DXA. A total of 22 fatty acids were identified in marrow and subcutaneous fat. Significant differences in fatty acid composition existed between marrow and subcutaneous fat as well as between marrow fat samples obtained from the proximal femur and proximal tibia. Other than cis-7-hexadecenoic acid [C16:1 (n = 9)] and docosanoic acid [C22:0], no difference in marrow fatty acid composition was evident between subject groups of varying BMD (normal, low bone mass, and osteoporosis). In conclusion, there exists a wide range of individual fatty acids in marrow fat. Marrow fatty acid composition differs from that of subcutaneous fat and varies between predominantly erythropoetic and fatty marrow sites. Other than cis-7-hexadecenoic acid [C16:1 (n = 9)] and docosanoic acid [C22:0], no difference in marrow fatty acid composition was evident between subjects of varying BMD.     

Differential expression of platelet-derived growth factor receptors in porcine fibroblasts cultured from skin and granulation tissue

This study investigated the biological response of fibroblasts cultured from uninjured skin and granulation tissue from different stages of healing wounds to the three isoforms of platelet-derived growth factor. Fibroblasts were derived by explant culture from the skin or the granulation tissue that formed within open mesh nylon Schilling-Hunt chambers (postoperative days 10, 20, 30, and 50) which had been implanted subcutaneously in the backs of domestic pigs. Cells were cultured under identical conditions in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Mitogenic activity was measured with (3)H-thymidine incorporation into DNA. Fibroblasts from normal skin responded equally well to all of the platelet-derived growth factor isoforms in the mitogenic assays. All of the wound fibroblasts exhibited a decreased response to platelet-derived growth factor compared with those from skin. Granulation tissue fibroblasts responded to platelet-derived growth factor BB, less to platelet-derived growth factor AB, and poorly to platelet-derived growth factor AA. These results correlated with a significantly decreased growth rate of fibroblasts in culture from both 30- and 50-day postsurgical wound tissue compared with normal skin. Western blot studies of cell membrane extracts showed that wound fibroblasts contained less than 20% as many platelet-derived growth factor-alpha receptors as found in fibroblasts cultured from normal skin. No significant difference in the amount of platelet-derived growth factor-beta receptor was detected. The decreases in platelet-derived growth factor-alpha receptors are sufficient to account for the diminished response of the wound fibroblasts to all platelet-derived growth factor isoforms and the differential loss of responsiveness to platelet-derived growth factor AA. These results show that fibroblasts derived from granulation tissue of pig skin wounds exhibit a decreased growth response to platelet-derived growth factor and a decreased growth rate in culture media as compared with fibroblasts derived from uninjured skin. How these differences may relate to the physiologic characteristics of normal and healing-impaired wounds is considered.     

Changes in matrix proteoglycans induced by insulin and fatty acids in hepatic cells may contribute to dyslipidemia of insulin resistance

Insulin resistance and type 2 diabetes are associated with elevated circulating levels of insulin, nonesterified fatty acids (NEFAs), and lipoprotein remnants. Extracellular matrix proteoglycan (PG) alterations are also common in macro- and microvascular complications of type 2 diabetes. In liver, extracellular heparan sulfate (HS) PGs contribute to the uptake of triglyceride-rich lipoprotein remnants. We found that HepG2 cells cultured with 10 or 50 nmol/l insulin or 300 micromol/l albumin-bound linoleic acid changed their PG secretion. The glycosaminoglycans (GAGs) of the secreted PGs from insulin-treated HepG2 cells were enriched in chondroitin sulfate (CS) PGs. In contrast, cells exposed to linoleic acid secreted PGs with decreased content of CS. Insulin caused a moderate increase in mRNA for versican (secreted CS PG), whereas linoleic acid markedly decreased mRNA for versican in HepG2 cells, as did the peroxisomal proliferator-activated receptor-alpha agonist bezafibrate. The effects of insulin or linoleic acid on syndecan 1, a cell surface HS PG, were similar to those on versican, but less pronounced. The livers of obese Zucker fa/fa rats, which are insulin-resistant and have high levels of insulin, NEFAs, and triglyceride-rich remnants, showed increased expression of CS PGs when compared with lean littermates. These changes in PG composition decreased the affinity of remnant beta-VLDL particles to PGs isolated from insulin-treated HepG2 cells and obese rat livers. The results indicated that insulin and NEFAs modulate the expression of PGs in hepatic cells. We speculate that in vivo this exchange of CS for HS may reduce the clearance of remnant beta-VLDLs and contribute to the dyslipidemia of insulin resistance.     

Phospholipid composition and fatty acid profiles of the phospholipids in bovine predentin

Summary When bovine dental pulp was removed from erupted and unerupted molars the odontoblasts remained attached to the predentin-dentin shell, from which the soft, partially calcified predentin layer was removed for lipid analysis.The phospholipid and fatty acid composition of phospholipids and neutral lipids were the same in undemineralized bovine predentin from both erupted and unerupted calf molars. The major phospholipids present were phosphatidylcholine (52–56% of the total phospholipids) and ethanolamine phosphoglycerides (22–23%). About 10% of the ethanolamine phosphoglycerides were plasmalogens. The major fatty acids observed were palmitic, stearic, oleic, linoleic, and arachidonic acids. The phospholipid composition of predentin was different from that of undemineralized dental pulp. Predentin contained a significantly higher percentage of phosphatidylserine and sphingomyelin than dental pulp. The fatty acid profiles of the individual phospholipids in the bovine predentin layer were also determined, and for the most part they were comparable to similar comparisons made for other tissues. Predentin sphingomyelin contained only about 6% of 24-carbon fatty acids, which is a relatively low amount compared to sphingomyelin in most other tissues.The fatty acid compositions of predentin neutral lipids and phospholipids were similar to the compositions of dental pulp lipids, but both types of predentin lipids contained more docosapolyenoic acids. The phospholipid and fatty acid compositions of predentin lipids were different from those in human and rat dentin which have been reported by others.     

Effect of serum on human bone marrow stromal cells: ex vivo expansion and in vivo bone formation

BACKGROUND: Bone marrow stromal cell (BMSC) transplantation may offer an efficacious method for the repair of bone defects. This approach has been developed using BMSCs expanded ex vivo in medium with fetal bovine serum (FBS). For clinical applications, however, contact of BMSCs with FBS should be minimized. We studied the effect of FBS substitutes on both human BMSC proliferation in vitro and subsequent bone formation in vivo. METHODS: BMSC proliferation was measured by colony forming efficiency (CFE) and by cell numbers at consecutive passages. Bone formation was studied in 6- to 8-week-old transplants of human BMSCs in immunocompromised mice. RESULTS: Medium with FBS was more effective in stimulating BMSC proliferation than medium with either human serum (HS) or rabbit serum (RS). Compared to bone formed by BMSCs cultured continuously with FBS, bone formed by cells cultured with HS, or with FBS switched to HS, was considerably less extensive, while bone formed by cells cultured with FBS switched to serum-free medium (SFM) was considerably more extensive. The increase in bone formation was due to neither the SFM components nor to the proliferation status of BMSCs prior to transplantation. CONCLUSIONS: Our data demonstrate that for ex vivo expansion of human BMSCs, medium with FBS remains most effective. However, incubation of human BMSCs in SFM prior to in vivo transplantation significantly stimulates subsequent bone formation. This finding increases the practicality of using culture-expanded BMSCs for autologous human transplantation and suggests the presence of osteogenic inhibitors in serum.