低强度脉冲超声波对细胞增殖及生物合成的影响

低强度脉冲超声波具有独特的生物学效应,在体外细胞及体内实验中研究发现能够调节细胞的增殖、促进胶原及非胶原蛋白的合成、影响细胞因子的分泌和诱导细胞分化作用,其在临床治疗活动中已经在骨折,放射性骨坏死的治疗中取得一定成效。本文综述了近年来在低强度脉冲超声波生物学效应研究的新进展。     

Low‐intensity pulsed ultrasound combination with induced pluripotent stem cells‐derived neural crest stem cells and growth differentiation factor 5 promotes sciatic nerve regeneration and functional recovery

The treatment of lengthy peripheral nerve defect is challenging in the field of nerve regeneration. Our previous studies have shown that low‐intensity pulsed ultrasound (LIPUS) could promote the proliferation, cell viability, and neural differentiation of induced pluripotent stem cells‐derived neural crest stem cells (iPSCs‐NCSCs) and improve the regeneration of damaged peripheral nerve. In this study, the mechanical signal transduction pathway of LIPUS promoting iPSCs‐NCSCs proliferation and differentiation was further explored, and the effects of LIPUS combined with iPSCs‐NCSCs, perfluorotributylamine (PFTBA), and growth differentiation factor 5 (GDF5) on the repair of peripheral nerve injury were evaluated. Results showed LIPUS may regulate the proliferation and differentiation of iPSCs‐NCSCs through FAK‐ERK1/2 signal pathway. PFTBA could supply sufficient oxygen to promote the viability of iPSCs‐NCSCs under 5% hypoxia culture condition and provide a favourable microenvironment for nerve regeneration. The addition of GDF5 could promote the neural differentiation of iPSCs‐NCSCs in vitro. LIPUS treatment of allogeneic decellularized nerve conduit containing iPSCs‐NCSCs, PFTBA, and GDF5 has very good effect on the repair of sciatic nerve injury. Taken together, these results provide functional evidence that LIPUS might be a useful tool to explore alternative approaches in the field of nerve regeneration.     

Adenine nucleotides stimulate migration in wounded cultures of kidney epithelial cells.

Adenine nucleotides speed structural and functional recovery when administered after experimental renal injury in the rat and stimulate proliferation of kidney epithelial cells. As cell migration is a component of renal regeneration after acute tubular necrosis, we have used an in vitro model of wound healing to study this process. High density, quiescent monkey kidney epithelial cultures were wounded by mechanically scraping away defined regions of the monolayer to simulate the effect of cell loss after tubular necrosis and the number of cells that migrated into the denuded area was counted. Migration was independent of cell proliferation. Provision of adenosine, adenine nucleotides, or cyclic AMP increased the number of migrating cells and accelerated repair of the wound. Other purine and pyrimidine nucleotides were not effective. Arginine-glycine-aspartic acid-serine peptide, which blocks the binding of extracellular fibronectin to its cell surface receptor, completely inhibited migration in the presence or absence of ADP. Very low concentrations of epidermal growth factor (K0.5 approximately 0.3 ng/ml) stimulated migration, whereas transforming growth factor-beta 2 was inhibitory (Ki approximately 0.2 ng/ml). Thus, adenosine and/or adenine nucleotides released from injured or dying renal cells, or administered exogenously, may stimulate surviving cells in the wounded nephron to migrate along the basement membrane, thereby rapidly restoring tubular structure and function.     

低强度脉冲超声波刺激对人骨髓基质细胞和骨膜细胞生物学效应的影响

目的研究低强度脉冲超声波（LIPUS）对体外分离培养的人骨髓基质细胞、骨膜细胞生物学效应的影响，探讨其促进骨折愈合的相关机制。方法采用辐射强度为30mW／cm^2的脉冲超声波分别作用于体外培养的人骨髓基质细胞、骨膜细胞（上述细胞根据LIPUS每天作用时间再分为5min／d组，10min／d组及20min／d组），并于不同时问点检测其对细胞生长、分化等主要相关指标的影响。结果各LIPUS实验组骨髓基质细胞、骨膜细胞DNA合成量与空白对照组比较，差异均无统计学意义（P〉0．05）；骨膜细胞经LIPUS作用后，其ALP活性、骨钙素分泌水平及钙结节形成均显著增强（P〈0．05），而骨髓基质细胞经LIPUS作用后．其细胞分化标志物均无明显改变（P〉0．05）。结论LIPUS能够促进人骨膜细胞向成骨细胞分化，这可能是其促进骨折愈合的相关生物学机制之一。     

Using Low-Intensity Pulsed Ultrasound to Improve Muscle Healing After Laceration Injury: An in vitro and in vivo Study

The purpose of this study was to determine whether low-intensity pulsed ultrasound (LIPUS) could enhance the regeneration of myofibers and shorten the healing time in injured muscle. NIH C2C12 cells, a well-known myoblastic cell line, are subclones derived from the mouse myoblast cell line established from normal adult C3H mouse leg muscle. The cells differentiate rapidly and produce extensive contracting myotubes expressing characteristic muscle proteins. We exposed C2C12 cells to LIPUS therapy using the EXOGEN 2000+ system ultrasound apparatus (Exogen Inc., Piscataway, NJ, USA) with a total treatment of 20 min every 24 h. At intervals of 2, 4, 6 and 8 days, cell growth was measured by the increase in cell number and western blot analysis of myogenin and actin. Forty mice (C57BL10J+/+) were divided into five groups of eight animals each and used in the published laceration injury model. The gastrocnemius muscle of the left leg was lacerated in all the animals. The control group (sham ultrasound) did not undergo LIPUS therapy. The ultrasound 7-, 14-, 21- and 28-day groups (only changing the number of days during which the ultrasound was applied to the injured muscle) were treated with LIPUS (20 min/day) for 7, 14, 21 and 28 consecutive days, respectively. All animals were sacrificed at 4 weeks after the injury. Evaluation methods included muscle regeneration and muscle contractile properties. LIPUS therapy produced a significantly higher proliferative rate and cell number at days 6 and 8 (p < 0.05). Densitometric evaluation revealed an increase in myogenin and actin proteins in cells treated with LIPUS in the 4-, 6- and 8-day groups. The regeneration of myofibers, fast-twitch and tetanus of LIPUS-treated muscles (21 and 28 days) was significantly greater relative to control muscles. There was no major strength difference between the normal noninjured muscle and the group treated with LIPUS for 28 days. In conclusion, this was the first experimental study to show that LIPUS therapy is able to enhance the regeneration of myofibers with better physiologic performance in injured mice muscles after laceration, especially prior to postoperative week 4. Findings of this study demonstrate a scientific basis for future clinical trials and establish an indication for LIPUS in enhancing muscle healing after laceration injury. (E-mail: chan512@adm.cgmh.org.tw)     

Low-Intensity Pulsed Ultrasound Promotes Chondrogenic Progenitor Cell Migration via Focal Adhesion Kinase Pathway

Low-intensity pulsed ultrasound (LIPUS) has been studied frequently for its beneficial effects on the repair of injured articular cartilage. We hypothesized that these effects are due to stimulation of chondrogenic progenitor cell (CPC) migration toward injured areas of cartilage through focal adhesion kinase (FAK) activation. CPC chemotaxis in bluntly injured osteochondral explants was examined by confocal microscopy, and migratory activity of cultured CPCs was measured in transwell and monolayer scratch assays. FAK activation by LIPUS was analyzed in cultured CPCs by Western blot. LIPUS effects were compared with the effects of two known chemotactic factors: N-formyl-methionyl-leucyl-phenylalanine (fMLF) and high-mobility group box 1 (HMGB1) protein. LIPUS significantly enhanced CPC migration on explants and in cell culture assays. Phosphorylation of FAK at the kinase domain (Tyr 576/577) was maximized by 5 min of exposure to LIPUS at a dose of 27.5 mW/cm² and frequency of 3.5 MHz. Treatment with fMLF, but not HMBG1, enhanced FAK activation to a degree similar to that of LIPUS, but neither fMLF nor HMGB1 enhanced the LIPUS effect. LIPUS-induced CPC migration was blocked by suppressing FAK phosphorylation with a Src family kinase inhibitor that blocks FAK phosphorylation. Our results imply that LIPUS might be used to promote cartilage healing by inducing the migration of CPCs to injured sites, which could delay or prevent the onset of post-traumatic osteoarthritis.     

低强度脉冲超声介导威灵仙干预兔膝关节软骨细胞增殖及Ⅱ型胶原和转化生长因子β1的表达

背景：研究显示威灵仙可促进软骨细胞增殖,维持和促进软骨细胞合成蛋白多糖和Ⅱ型胶原;低强度脉冲超声能有效促进软骨细胞增殖、提高细胞膜的通透性,促进药物或基因的传递,从而提高药物的生物学效应。目的：观察低强度脉冲超声介导威灵仙对体外培养的兔膝关节软骨细胞增殖、Ⅱ型胶原及转化生长因子β1表达的影响,探讨低强度脉冲超声介导威灵仙在软骨损伤修复中的作用及机制。方法：体外分离培养兔膝关节软骨细胞,取处于对数生长期的2代细胞,随机分为4组：对照组,低强度脉冲超声组,威灵仙组,威灵仙＋低强度脉冲超声组,干预3 d后分别采用CCK-8比色法检测软骨细胞增殖情况,细胞化学染色法分析Ⅱ型胶原分泌情况及Western blot检测转化生长因子β1的表达情况。结果与结论：干预培养3 d后,与对照组比较,其他3组细胞数量均显著增加（P〈0.05）,威灵仙＋低强度脉冲超声组细胞数量最多;威灵仙＋低强度脉冲超声组软骨细胞Ⅱ型胶原表达面积、吸光度值均显著大于对照组（P〈0.05）,且其差值大于低强度脉冲超声组、威灵仙组与对照组差值之和;威灵仙＋低强度脉冲超声组转化生长因子β1表达水平显著,其相对灰度值显著高于其他各组（P〈0.05）。结果提示低强度脉冲超声、威灵仙均可促进体外培养兔膝关节软骨细胞增殖及Ⅱ型胶原、转化生长因子β1的分泌,二者联用具有一定的协同作用。     

低强度脉冲超声治疗平滑肌疾病及其作用机制研究进展

低强度脉冲超声(LIPUS)强度远低于传统超声,通常以低强度、脉冲式模式给予靶目标以热效应、空化效应及机械效应等无创性物理及生物刺激,可促进骨折愈合,刺激肌腱、韧带和软骨等软组织再生,并抑制炎症反应,促进骨骼肌修复.本文对LIPUS治疗平滑肌疾病及其作用机制研究进展进行综述.     

Osteogenic effects of low-intensity pulsed ultrasound, extracorporeal shockwaves and their combination - an in vitro comparative study on human periosteal cells

Our previous studies have shown that on human periosteal cells, low-intensity pulsed ultrasound (LIPUS) has an immediate stimulatory effect whereas extracorporeal shockwaves (ESW) have an delayed stimulatory effect. Therefore, we hypothesized that a combined ESW and LIPUS treatment might provide additive or synergistic effects on periosteal cells, by using ESW to trigger a biological activity while using LIPUS to maintain the stimulated activity. Human periosteal cells were subjected to a single session of ESW treatment on day 0 and/or daily LIPUS treatments or no treatment (control). The cell viability, proliferation, and alkaline phosphatase activity on day 6 and day 18 as well as matrix mineralization on day 35 were measured. Results revealed that LIPUS alone had early positive effects on the activities on day 6 only. In contrast, ESW alone had an early destructive effect but exerted delayed stimulatory effects on the cellular activities on day 18. The combined treatment of ESW plus LIPUS produced effects that were comparable to the ESW treatment alone. Although these findings suggest that ESW and LIPUS stimulate the periosteal cells in two different ways and at different times, their additive or synergistic effects could not be proven.     

The dual delivery of KGF and bFGF by collagen membrane to promote skin wound healing

The major challenges associated with skin regeneration can include hindered vascularization and an insufficient degree of epithelization. In view of the complexity of these processes and the control signals on which they depend, one possible solution to these limitations could be simulating normal skin development and wound repair via the exogenous delivery of multiple cytokines. Here, we report the use of keratinocyte growth factor (KGF or FGF‐7) and basic fibroblast growth factor (bFGF or FGF‐2) released chemically modified collagen membranes to facilitate skin wound healing. The results from in vitro studies confirmed that this system resulted in higher cellular proliferation and faster cell migration. After transplanting the biomaterial onto an excisional wound healing model, the dual growth factor group, compared with the single growth factor groups and empty control group, showed more highly developed vascular networks and organized epidermal regeneration in the wounds. As a consequence, this experimental group showed mature epidermal coverage. Overall, this novel approach of releasing growth factors from a collagen membrane opens new avenues for fulfilling unmet clinical needs for wound care.     

Non-viral liposomal keratinocyte growth factor (KGF) cDNA gene transfer improves dermal and epidermal regeneration through stimulation of epithelial and mesenchymal factors

Keratinocyte growth factor (KGF) stimulates epithelial cell differentiation and proliferation, which are of major importance for wound healing. Local protein administration, however, has been shown to be ineffective due to enzymes and proteases in the wound fluid. We hypothesized that delivering KGF as a non-viral liposomal cDNA gene complex is a new approach that would effectively enhance dermal and epidermal regeneration. Twenty-two rats were given an acute wound and divided into two groups to receive weekly subcutaneous injections of liposomes plus the LacZ gene (0.2 microg, vehicle), or liposomes plus the KGF cDNA (2.2 microg) and LacZ cDNA (0.2 microg). Transfection was confirmed by histochemical assays for beta-galactosidase. Planimetry, histological and immunohistochemical techniques were used to determine protein expression, dermal and epidermal regeneration. Transfection and subsequent KGF expression was found in diving cells in the granulation tissue. Epidermal regeneration was improved by 170% in rats receiving the KGF cDNA constructs by exhibiting the most rapid area and linear wound re-epithelialization, P < 0.0001. KGF improved epidermal cell net balance by increasing skin cell proliferation and decreasing skin cell apoptosis, P < 0.0001. Dermal regeneration was further improved in KGF cDNA treated animals by an increased collagen deposition and morphology, P < 0.0001. KGF cDNA increased neo-vascularization and concomitant VEGF concentrations when compared with vehicle, P < 0.01. KGF cDNA did not only stimulate epithelial cells, but also mesenchymal cells through increases in IGF-I concentration, P < 0.005. Liposomes containing the KGF cDNA gene constructs were effective in improving epidermal and dermal regeneration. KGF gene transfer to acute wounds may represent a new therapeutic strategy to enhance wound healing.     

低强度超声对离体大鼠膀胱平滑肌收缩的影响

目的 观察低强度脉冲超声(LIPUS)对离体大鼠膀胱平滑肌收缩的影响。方法 将32只SD雌性大鼠的32段离体膀胱肌条随机分为超声辐照组、超声辐照+尼莫地平组、超声辐照+低分子肝素组及对照组,每组各8段。采用BL-410F生物机能实验系统测量并记录超声辐照组、超声辐照+尼莫地平组、超声辐照+低分子肝素组在LIPUS辐照前、后的收缩曲线,并比较3组膀胱平滑肌条收缩频率、幅度。对超声辐照组LIPUS辐照后和对照组膀胱平滑肌进行组织病理学观察。结果 与LIPUS辐照前比较,辐照后超声辐照组的收缩频率和幅度均明显增加(P均<0.05),超声辐照+尼莫地平组的收缩频率和幅度均明显降低(P均<0.05),超声辐照+低分子肝素组的收缩频率和幅度均无明显变化(P均>0.05)。组织病理学显示超声辐照组膀胱平滑肌细胞与对照组比较无明显差异。结论 LIPUS辐照可激活膀胱平滑肌细胞膜L-型钙通道,继而引起膀胱平滑肌的收缩增强。     

Growth Modulation by Stimulating the Growth Plate: A Pilot Study

This study investigates the ability of low-intensity pulsed ultrasound (LIPUS) or direct injection of recombinant growth hormone (rGH) to stimulate local growth of long bones. In a randomized controlled animal trial, healthy immature rabbits were allocated to 1 of the following 4 conditions: epiphyseal rGH periosteal injection, transdermal LIPUS, saline periosteal injection, or no treatment. New bone deposition was labeled with calcein at days 1 and 18, and microscopic measurements of growth were conducted by blinded observers. Statistically significant differences in growth were observed between the LIPUS and rGH stimulated legs compared with contralateral control legs (35% p = 0.04 and 41% p = 0.04, respectively); whereas no difference was observed between the 4 control groups (p = 0.37). There was no evidence of physeal bar formation, suggesting that direct injection of rGH and application of LIPUS around the distal femoral physis in rabbits may have a positive effect on microscopic growth without short-term adverse sequelae.     

Low-intensity pulsed ultrasound accelerated callus formation, angiogenesis and callus remodeling in osteoporotic fracture healing

Osteoporotic fracture is a critical medico-social challenge leading to burdens in health care costs and hospital bed stays. Low-intensity pulsed ultrasound (LIPUS) was reported to accelerate normal fracture; however, its effect on osteoporotic fracture has not been previously addressed. We hypothesize that LIPUS can accelerate osteoporotic fracture healing and up-regulate the expression in the osteogenesis-, remodeling- and angiogenesis-related genes. Ovariectomy-induced osteoporotic fracture rat model was used to investigate the effects of LIPUS. Fractured rats were assigned to LIPUS or control group and healing was assessed by gene expression quantification, radiographic callus morphometry and histomorphometry. In the LIPUS group, Col-1 and bone morphogenetic protein-2 were up-regulated at earlier time points of week 2 to week 4 post-fracture; vascular endothelial growth factor was found to be up-regulated at week 4 to week 8; osteoprotegerin was up-regulated at week 2 post-fracture, followed by the surge of RANKL expression. Callus width and area measurements showed higher callus formation at weeks 2-4 in the LIPUS group and more rapid drop at weeks 6-8. Histomorphometry showed enhanced endochondral ossification in the callus at weeks 2-4, and lower at week 8. We conclude that LIPUS can accelerate osteoporotic fracture healing by enhancing callus formation, angiogenesis and callus remodeling. (E-mail: louis@ort.cuhk.edu.hk)     

Spleen tyrosine kinase mediates BEAS-2B cell migration and proliferation and human rhinovirus-induced expression of vascular endothelial growth factor and interleukin-8

Spleen tyrosine kinase (Syk) is an immunoregulatory tyrosine kinase that was identified originally in leukocytes. It is a key regulator of innate immunity as well as hematopoietic cell differentiation and proliferation. A role for Syk in regulating normal cellular functions in nonhematopoietic cells is increasingly recognized. We have shown previously robust Syk expression in airway epithelium, where it regulates the early inflammatory response to human rhinovirus (HRV) infections, and HRV cell entry by clathrin-mediated endocytosis. To test the hypothesis that Syk plays a role in modulating airway epithelial cell proliferation, migration, and production of vascular endothelial growth factor and interleukin-8, we studied the BEAS-2B human bronchial epithelial cell line and primary human airway epithelia from normal and asthmatic donors using Syk-specific pharmacologic inhibitors and small interfering RNA. Using an in vitro "wounding" model, we demonstrated significant impairment of "wound" closure after treatment with the Syk inhibitors N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406) and 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]-nicotinamide dihydrochloride (BAY61-3606), overexpression of the kinase-inactive Syk(K396R) mutant, and Syk knockdown by small interfering RNA. HRV infection also impaired wound healing, an effect that was partly Syk-dependent because wound healing was impaired further when HRV infection occurred in the presence of Syk inhibition. Further investigation of potential regulatory mechanisms revealed that inhibition of Syk suppressed HRV-induced vascular endothelial growth factor expression while promoting the activation of caspase-3, a mediator of epithelial cell apoptosis. Together, these results indicate that Syk plays a role in promoting epithelial cell proliferation and migration, while mitigating the effects of apoptosis.     

Is low-intensity pulsed ultrasound effective for revitalizing a severely necrotic small bone? An experimental rabbit model

Previously, we successfully applied a new method composed of drilling, bone marrow transplantation (BMT), external fixation and low-intensity pulsed ultrasound (LIPUS) for the clinical treatment of Kienböck’s disease. The purpose of this study was to investigate whether bone regeneration can be induced by LIPUS and/or multiple drilling and/or BMT within a severely necrotic small-bone rabbit model. Eighteen rabbits were divided into three groups (BMT, drilling and control) and LIPUS stimulation was introduced daily for 8 weeks post-transplantation. Next, 12 additional rabbits were produced for the BMT group and LIPUS stimulation was introduced daily for 4 and 12 weeks (n = 6 for each). Histopathologically, new bone formations were rarely observed in the drilling and control groups. In the BMT group, the mineralizing surface areas of LIPUS(+) showed a significant increase compared with LIPUS(−) for 8 weeks. LIPUS treatment alone did not accelerate the revitalization of necrotic bones. However, LIPUS combined with BMT tended to promote new bone formation.     

Acceleration of Bone Defect Healing and Regeneration by Low-Intensity Ultrasound Radiation Force in a Rat Tibial Model

The objective was to evaluate the effect of low-intensity pulsed ultrasound (LIPUS)-induced acoustic radiation force on trabecular bone defect repair and healing in a rat tibial model. A uniform surgical defect, 3.5 mm in diameter, was generated in the proximal bilateral tibial region of rats (N?=?20). LIPUS was applied to the defects in the left tibia for 20 min every day for 2 wk. Contralateral defects in the right tibia served as a control without active LIPUS treatment. The micro-computed tomography data revealed that LIPUS-treated tibia exhibited higher bone volume/total volume, connectivity density, trabecular number, and bone mineral density and significantly lower trabecular separation. Histomorphometry analysis indicated a similar trend. Mechanical testing data revealed that LIPUS treatment significantly increased bone stiffness relative to that of the control group. Short-term (2-wk) LIPUS therapy initiated trabecular bone repair and regeneration in large trabecular bone defects, whereas cortical bone remained in the initial non-mineralization stage.     

Hepatocyte growth factor is the most potent endogenous stimulant of rabbit gastric epithelial cell proliferation and migration in primary culture.

Various growth factors are suggested to be involved in gastric mucosal repair. Our previous studies have shown that exogenous hepatocyte growth factor (HGF) has a proliferative effect on gastric epithelial cells. In the present study, comparison of the maximum proliferative effects and the optimum concentrations of several growth factors revealed that HGF was the most potent mitogen for gastric epithelial cells, as is the case for hepatocytes. Restitution of gastric epithelial cell monolayers was assessed using a round wound restitution model. HGF was the most effective agent for facilitating gastric epithelial restitution among those tested. A binding assay revealed specific binding of HGF to its receptor on gastric epithelial cells. Northern blot analysis confirmed the expression of specific HGF receptor mRNA (c-met) by gastric epithelial cells but not by gastric fibroblasts. To investigate endogenous HGF production, we determined the effect of gastric fibroblast-conditioned medium on epithelial proliferation and restitution. The conditioned medium produced similar effects to HGF and its activity was neutralized by an anti-HGF antibody. In addition, expression of HGF mRNA was detected in gastric fibroblasts but not in gastric epithelial cells. Our immunohistochemical study confirmed these in vitro data by means of demonstrating the existence and localization of HGF at human native gastric mucosa. HGF was localized at fibroblasts under the epithelial cell layer around gastric ulcers. These results suggest that HGF may be a potent endogenous promotor of gastric epithelial cell proliferation and migration, and may contribute to gastric mucosal repair through a paracrine mechanism.     

Effects of exogenous zinc supplementation on intestinal epithelial repair in vitro

BACKGROUND: Substitution of zinc modulates antioxidant capabilities within the intestinal mucosa and improves intestinal wound healing in zinc-deficient patients with inflammatory bowel diseases. The aim of this study was to characterize the modulating effects of zinc on intestinal epithelial cell function in vitro. MATERIALS AND METHODS: The effects of zinc on intestinal epithelial cell morphology were assessed by phase contrast and transmission electron microscopy using the non-transformed small intestinal epithelial cell line IEC-6. Zinc-induced apoptosis was assessed by DNA fragmentation analysis, lactate dehydrogluase (LDH) release and flow cytometry with propidium iodine staining. Furthermore, the effects of zinc on IEC-6 cell proliferation were assessed using a colorimetric thiazolyl blue (MTT) assay and on IEC-6 cell restitution using an in vitro wounding model. RESULTS: Physiological concentrations of zinc (25 microM) did not significantly alter the morphological appearance of IEC-6 cells. However, a 10-fold higher dose of zinc (250 microM) induced epithelial cell rounding, loss of adherence and apoptotic characteristics. While physiological zinc concentrations (< 100 microM) did not induce apoptosis, supraphysiological zinc concentrations (> 100 microM) caused apoptosis. Physiological concentrations of zinc (6.25-50 microM) had no significant effect on intestinal epithelial cell proliferation. In contrast, physiological concentrations of zinc (12.5-50 microM) significantly enhanced epithelial cell restitution through a transforming growth factor-beta (TGFbeta)-independent mechanism. Simultaneous addition of TGFbeta and zinc resulted in an additive stimulation of IEC-6 cell restitution. CONCLUSION: Zinc may promote intestinal epithelial wound healing by enhancement of epithelial cell restitution, the initial step of epithelial wound healing. Zinc supplementation may improve epithelial repair; however, excessive amounts of zinc may cause tissue injury and impair epithelial wound healing.