Collagen glycosylation in human granulation tissue and scar

Some biochemical characteristics of collagen extracted from granulation tissue were studied and compared with those of normal skin and scar. By using electrophoretic techniques the type III collagen content was confirmed to be significantly greater in granulation tissue and lower in scar with respect to normal skin. The chromatographic determination of hydroxylysine (Hyl) glycosides in collagen extracted from granulation tissue showed a significant increase in both the degree of Hyl glycosylation and in the di-/monoglycoside ratio, while both parameters turned out to be lower in scar. These data suggest that the degree of Hyl glycosylation and the di-/monoglycoside ratio could represent an index of the degree of collagen fiber maturation.     

Circulatory and mechanical response of skin to loading

We investigated the tolerance of skin to mechanical loading over the tibia and over the tibialis anterior muscle in 12 normal volunteers. Surface load, subcutaneous tissue pressure, skin deformation under load, and transcutaneous partial pressure of oxygen (TcPO2) were simultaneously monitored. The skin over bone showed a significantly stiffer load deformation relationship than the skin over muscle (p less than 0.001). The displacement required to reduce TcPO2 to 0 over bone, 1.1 +/- 0.3 mm (mean +/- standard deviation), was significantly less than that required over tibialis anterior muscle, 5.4 +/- 1.1 mm (p less than 0.001). The applied pressure required to reduce TcPO2 to 0 was significantly greater for skin over muscle (71 +/- 16 mm Hg) than for skin over bone (42 +/- 8 mm Hg) (p less than 0.001). However, the subcutaneous pressure required to reduce TcPO2 to 0 was not significantly different for skin over muscle (36 +/- 11 mm Hg) than for skin over bone (28 +/- 10 mm Hg) (p greater than 0.05). Our results indicate that skin over muscle tolerates greater locally applied loads and deformations because the pressure is lower within the tissue than when similar loads and deformation are applied to skin over bone. Cutaneous perfusion, as indicated by TcPO2, seems to be linked more closely to the subcutaneous tissue pressure than to the surface load or deformations. These results provide some data for predicting mechanical and physiologic response to locally applied loads such as those that may be encountered in prosthetic wear.     

Adaptation of the dermal collagen structure of human skin and scar tissue in response to stretch: An experimental study

Surgeons are often faced with large defects that are difficult to close. Stretching adjacent skin can facilitate wound closure. In clinical practice, intraoperative stretching is performed in a cyclical or continuous fashion. However, exact mechanisms of tissue adaptation to stretch remain unclear. Therefore, we investigated collagen and elastin orientation and morphology of stretched and nonstretched healthy skin and scars. Tissue samples were stretched, fixed in stretched‐out position, and processed for histology. Objective methods were used to quantify the collagen orientation index (COI), bundle thickness, and bundle spacing. Also sections were analyzed for elastin orientation and quantity. Significantly more parallel aligned collagen bundles were found after cyclical (COI = 0.57) and continuous stretch (COI = 0.57) compared with nonstretched skin (COI = 0.40). Similarly, more parallel aligned elastin was found after stretch. Also, significantly thicker collagen bundles and more bundle spacing were found after stretch. For stretched scars, significantly more parallel aligned collagen was found (COI = 0.61) compared with nonstretched scars (COI = 0.49). In conclusion, both elastin and collagen realign in a parallel fashion in response to stretch. For healthy skin, thicker bundles and more space between the bundles were found. Rapid changes in extension, alignment, and collagen morphology appear to be the underlying mechanisms of adaptation to stretching.     

体外培养人增生性瘢痕成纤维细胞胶原合成及结缔组织生长因子的表达

目的　探讨结缔组织生长因子在人增生性瘢痕发病机制中的作用。方法　体外培养人正常皮肤和增生性瘢痕成纤维细胞 ,通过H3 脯氨酸掺入法检测细胞胶原合成 ,通过免疫细胞化学染色和逆转录聚合酶链反应检测细胞结缔组织生长因子蛋白质和mRNA的表达。结果　和正常皮肤成纤维细胞相比 ,增生性瘢痕成纤维细胞的胶原合成和结缔组织生长因子蛋白质及mRNA的表达均显著增高 (P <0 0 1)。结论　结缔组织生长因子可能在增生性瘢痕纤维化过程中发挥重要促进作用。     

老化皮肤的胶原与弹性组织变化研究

目的：探讨光曝露情况及年龄因素对皮肤中弹性组织、Ⅰ/Ⅲ型胶原及基底膜Ⅳ型胶原的影响,进而探索皱纹形成的组织学原因。方法：对80例取自不同年龄志愿者的曝光部位（眶周/前额、面颊）及非曝光部位（腹部/臀部）的皮肤组织进行HE染色、Weigert染色、苦味酸天狼猩红染色及免疫组化处理,采用显微形态观察和半定量分析。结果：各部位皮肤基底膜内Ⅳ型胶原的含量无差异;随着曝光时间增加,前额/眶周皮肤中Ⅰ型胶原减少率最大,弹性变性物质增加率最大。结论：前额/眶周皮肤的胶原显著减少和弹性变性物质明显增生,是该部位皮肤皱纹早发的重要原因。     

Histamine and 5-hydroxytryptamine in human scar tissue

Patients with unstable scars in some areas of the body complain of local itchiness and pain, which may be caused by histamine and serotonin. Therefore, I measured these substances in scar tissue in various conditions taken at the time of operation. The histamine value was determined by the amount of carbon dioxide produced in histidine decarboxylase activity (HDCA); the measurement of 5-hydroxytryptamine (5-HT) was done by the Ninhydrin method. High values of HDCA were usually found in skin flaps and scar tissue around pressure sores. 5-HT activity reached a higher level in unstable scars than in stable scars.     

Bone response to mechanical loading in adult rats with collagen-induced arthritis

To elucidate the effects of inflammation on the response of bone to mechanical stress, we performed experiments using a rat with collagen-induced arthritis (CIA) model. Six-month-old female Wistar rats were used in the experiment. Bovine type II collagen sensitization and additional sensitization after 1 week were preformed in all CIA groups. Loads were applied using a four-point bending device. The right tibia was loaded in both CIA and control (CONT) groups at 35 N (low groups), 40 N (medium groups), or 47 N (high groups) for 36 cycles at 2 Hz three times per week for 3 weeks. Histomorphometrical data were collected from the periosteal and endosteal surfaces of the tibia in all rats. The tibia periosteal surface was subdivided into lateral and medial surfaces. Formation surface (FS), mineral apposition rate (MAR) and bone formation rate (BFR) were calculated. At lateral surface of periosteal surface, all three parameters showed significant differences between the loaded and nonloaded tibiae. All these parameters were significantly lower in CIA groups than in CONT groups, and interaction was seen between applied loading and CIA. There was a significant correlation between peak strain and the right-left difference of FS in the CONT groups. At medial surface of periosteal surface, there were force-related increase in FS, MAR, and BFR on the loaded side in both CIA and CONT groups, except MAR in the CONT group. All three parameters showed significant differences between the loaded and nonloaded tibiae. At endocortical surface, force-related increase was observed only in FS on the loaded side in CONT groups, and FS was significantly higher on the loaded side than the nonloaded side. CIA lowered all three parameters significantly. We examined the response to mechanical loading on the tibia in untreated CONT rats and rats with CIA by bone histomorphometry, and found that arthritis suppressed bone formation induced by mechanical loading.     

Reduced expression of BMP-3 due to mechanical loading: a link between mechanical stimuli and tissue differentiation

Mechanical signaling and BMP expression appear to be involved in controlling the differentiation of cartilage in fracture repair, but the connection between mechanics and BMP signaling is not known. In this study of rats, we used a bone chamber to see how BMP gene expression was changed by a mechanical loading regime that induces cartilage formation in this model. We compared the still undifferentiated tissue in loaded and unloaded chambers in the same rat regarding the expression of TGFbeta-1, BMP-2, 3, 4, 5, 6, 7, CDMP-1, 2 and ALK-2 and 3 by using RT-PCR normalized against GAPDH. We found expression of TGFbeta-1, BMP-2 and 4 in all specimens, and BMP 5-7 and CDMPs in none. 1 week after loading started, BMP-3 was strongly expressed in the unloaded control specimens in 7 of 8 animals, but detectable in only I of the contralateral loaded ones. After 2 weeks of loading, the BMP-3 expression pattern was less clear, but with both time groups taken together, there was still less BMP-3 expression on the loaded side in 9 rats, more in 1 and no difference in 5 (p = 0.01). ALK-2 at 1 week was expressed in all specimens expressing BMP-3 and in none of the others. At 2 weeks, ALK-2 was expressed in all specimens. Thus, a loading regime, known to induce cartilage in this model, caused down-regulation of BMP-3 and ALK-2. The results are consistent with the view that BMP-3 inhibits differentiation, as recently described. This role appears to be linked to the ALK-2 receptor. Most importantly, the results indicate a link between mechanical signaling and BMP expression such that mechanically-induced down-regulation of the inhibiting BMP-3 enabled the induction of cartilage.     

Reduced expression of BMP-3 due to mechanical loading: A link between mechanical stimuli and tissue differentiation

Mechanical signaling and BMP expression appear to be involved in controlling the differentiation of cartilage in fracture repair, but the connection between mechanics and BMP signaling is not known. In this study of rats, we used a bone chamber to see how BMP gene expression was changed by a mechanical loading regime that induces cartilage formation in this model. We compared the still undifferentiated tissue in loaded and unloaded chambers in the same rat regarding the expression of TGFß-1, BMP-2, 3, 4, 5, 6, 7, CDMP-1, 2 and ALK-2 and 3 by using RT-PCR normalized against GAPDH. We found expression of TGFß-1, BMP-2 and 4 in all specimens, and BMP 5-7 and CDMPs in none. 1 week after loading started, BMP-3 was strongly expressed in the unloaded control specimens in 7 of 8 animals, but detectable in only 1 of the contralateral loaded ones. After 2 weeks of loading, the BMP-3 expression pattern was less clear, but with both time groups taken together, there was still less BMP-3 expression on the loaded side in 9 rats, more in 1 and no difference in 5 (p = 0.01). ALK-2 at 1 week was expressed in all specimens expressing BMP-3 and in none of the others. At 2 weeks, ALK-2 was expressed in all specimens. Thus, a loading regime, known to induce cartilage in this model, caused down-regulation of BMP-3 and ALK-2. The results are consistent with the view that BMP-3 inhibits differentiation, as recently described. This role appears to be linked to the ALK-2 receptor. Most importantly, the results indicate a link between mechanical signaling and BMP expression such that me chanically-induced down-regulation of the inhibiting BMP-3 enabled the induction of cartilage.     

Proteoglycan synthesis in human skin and burn scar explant cultures

The synthesis of proteoglycans (PG) by normal human skin, and normal and hypertrophic scars were compared using tissue explants in culture. Newly synthesized PG were labelled with [³⁵S]Na₂SO⁴. Significant differences were found in the proportion of [³⁵S]-radio-labelled incorporation of PG in the tissue and accumulation of [³⁵S]PG in culture medium in the different tissues. The rate of PG biosynthesis in all three tissue types occurred in two phases. There was an initial phase of PG synthesis occurring at 0–3 h and a later phase that occurred at 3–18 h [³⁵S]-labelled PG were isolated and characterized by Sepharose CL-6B chromatography and cellulose acetate electrophoresis. The results showed that the hypertrophic scar tissue and its culture medium contained higher proportions of dermatan sulphate (DS), chondroitin sulphate (CS) and DS' PG than the normal skin fractions. These results suggest that abnormal scarring is related to a change in the level of PG synthesis during the burn injury repair process.     

Biomechanical behavior of scar tissue and uninjured skin in a porcine model

A new method to test axial and transverse tensile properties of skin was developed to improve our understanding of skin mechanical behavior, and how it changes following injury and formation of a scar. Skin tissue was evaluated at 70 days following full-thickness wounding in juvenile female pigs ( N =14). Samples were taken in the axial (cranial–caudal) and transverse (dorsal–ventral) directions, for both scar tissue and uninjured skin, and were evaluated mechanically in vitro using a protocol of stress relaxation followed by tensile failure. Uninjured skin was more compliant, with a larger toe-in region, and faster load relaxation, in the axial direction than the transverse. Such directional differences were not present in high-load responses, such as linear stiffness or failure properties. When compared with uninjured skin, scars displayed a similar linear stiffness, with considerably reduced failure properties, and reduced low-load compliance. Scars showed no directional differences in low-load behavior, viscous response, or failure properties. These findings suggest morphological changes that may occur with injury that are consistent with the viscoelastic and directional changes observed experimentally. This improved understanding of how injury affects skin biomechanical function provides valuable information necessary for the design of successful grafting procedures and tissue-engineered skin replacements.     

力学刺激对关节软骨基质代谢的影响

关节软骨主要由软骨细胞和细胞外基质组成,软骨细胞能够感受力学环境的变化而不断调整细胞外基质的代谢活动.适当的力学刺激会促进软骨细胞外基质的代谢,不适当的力学刺激有时不但会抑制细胞外基质的代谢,而且可能引起软骨细胞的变形或破坏而导致各种骨骼疾病的发生.正常的关节软骨一直处于静态压力与动态压力交替活动的力学环境中,体外构建工程化软骨过程中必须考虑这些力学因素,选择适当的力学刺激(力的作用方式、大小、频率、持续时间等)作用于软骨基质,使之形成具有体内软骨的力学特性.认识和加深理解力学刺激的作用机制,也有助于临床骨科诊疗观念和手段的发展与进步.该文就软骨基质的功能、力学刺激对软骨基质代谢的影响及在关节软骨组织工程中的应用展望,作一综述.     

Low-dose estrogen treatment suppresses periosteal bone formation in response to mechanical loading

Estrogen and exercise influence cortical bone formation. Both affect bone during growth, but with complex interactions. We hypothesized that estrogen reduces the osteogenic response caused by exercise at the periosteal surface of bone, while it enhances bone formation on the endocortical surface. To test our hypothesis, 16 young (8 weeks old) male Sprague-Dawley rats were randomized into two groups: (1) low-dose 17- ethynylestradiol treatment + bone loading (EE₂) or (2) vehicle-treated + bone loading (vehicle). We applied controlled loading to the right ulna at a peak force of 17 N, 2 min/day, 3 days/week for 5 weeks to simulate exercise. The left nonloaded ulna served as an internal control for loading. Mechanical loading increased cortical area (7.7%) and bone mineral content (8%) in the vehicle-treated group (P < 0.05) but only slightly increased cortical area in the EE₂ group (P = 0.08). Histomorphometry showed 1 week of mechanical loading increased periosteal bone formation rate by 29% in the vehicle group and this response was reduced (P < 0.05) to only 15% in the EE₂ group. At the endocortical surface, there were no differences in the loading response between the vehicle and EE₂-treated groups. We conclude low-dose EE₂ suppresses the mechanical loading response on the periosteal surface of long bones, but had no effect on the loading response at the endocortical bone surface in growing male rats.     

Remodeling and repair of orthopedic tissue: role of mechanical loading and biologics

Orthopedic tissues respond to mechanical loads to maintain normal homeostasis and in response to injury. As the body of work on this continues to grow, it is important to synthesize the recent studies across tissues and specialties with one another and with past studies. Hence, this review highlights the knowledge gained since 2000, with only few exceptions, concerning the effects of mechanical load and biologics on remodeling and repair of orthopedic tissue.     

Androgen receptor disruption increases the osteogenic response to mechanical loading in male mice

In female mice, estrogen receptor‐alpha (ERα) mediates the anabolic response of bone to mechanical loading. Whether ERα plays a similar role in the male skeleton and to what extent androgens and androgen receptor (AR) affect this response in males remain unaddressed. Therefore, we studied the adaptive response of in vivo ulna loading in AR‐ERα knockout (KO) mice and corresponding male and female single KO and wild‐type (WT) littermates using dynamic histomorphometry and immunohistochemistry. Additionally, cultured bone cells from WT and AR KO mice were subjected to mechanical loading by pulsating fluid flow in the presence or absence of testosterone. In contrast with female mice, ERα inactivation in male mice had no effect on the response to loading. Interestingly, loading induced significantly more periosteal bone formation in AR KO (+320%) and AR‐ERα KO mice (+256%) compared with male WT mice (+114%) and had a stronger inhibitory effect on SOST/sclerostin expression in AR KO versus WT mice. In accordance, the fluid flow‐induced nitric oxide production was higher in the absence of testosterone in bone cells from WT but not AR KO mice. In conclusion, AR but not ERα activation limits the osteogenic response to loading in male mice possibly via an effect on WNT signaling. © 2010 American Society for Bone and Mineral Research     

Lefty蛋白在人胚胎皮肤、成人正常皮肤及增生性瘢痕中的表达

目的 了解Lefty蛋白在人胚胎皮肤、成人正常皮肤及增生性瘢痕(HS)组织中的表达,探讨其在HS形成中的作用及与胚胎无瘢痕愈合的关系. 方法 留取人胚胎皮肤、成人正常皮肤及HS标本,制成冰冻切片,行免疫荧光染色.激光共聚焦显微镜下观察各标本中成纤维细胞形态及Lefty蛋白的表达,用阳性细胞率作为表达量. 结果 人胚胎皮肤成纤维细胞呈梭形,胞核呈椭圆形或梭形,排列规则;成人正常皮肤及HS组织成纤维细胞呈长梭形,胞核呈梭形或星形,前者排列较规则,而后者排列不规则.HS组织中Lefty蛋白阳性细胞率为15.38%,低于成人正常皮肤(67.92%)和人胚胎皮肤(81.67%,P＜0.01);而成人正常皮肤阳性细胞率也低于人胚胎皮肤(P＜0.05). 结论 Lefty蛋白可能对瘢痕的形成有抑制作用,其高表达可能与胚胎无瘢痕愈合相关.     

Concentration of orally administered antimicrobial agent in burn scar tissue, granulation tissue, normal skin and serum

Tissue and serum concentrations of orally administered ofloxacin were measured using high performance liquid chromatography. From 56 patients who received 200 mg of ofloxacin prior to surgery, 103 specimens including 48 of serum, 32 of scar tissue, 18 of normal skin and five of granulation tissue were harvested between 2 and 3 h after administration. The ofloxacin values were 1798.6 ± 1125.5 ng/g in the granulation tissue followed by 1525.5 ± 1002.7 ng/ml in the serum, 1450.0 ± 1011.3 ng/g in the scar tissue and 1092.8 ± 593.3 ng/g in normal skin. There was no statistically significant difference between the ofloxacin concentrations in those four specimens by ANOVA test.