Three dimensional microstructural network of elastin,collagen, and cells in Achilles tendons

Similar to most biological tissues, the biomechanical, and functional characteristics of the Achilles tendon are closely related to its composition and microstructure. It is commonly reported that type I collagen is the predominant component of tendons and is mainly responsible for the tissue's function. Although elastin has been found in varying proportions in other connective tissues, previous studies report that tendons contain very small quantities of elastin. However, the morphology and the microstructural relationship among the elastic fibres, collagen, and cells in tendon tissue have not been well examined. We hypothesize the elastic fibres, as another fibrillar component in the extracellular matrix, have a unique role in mechanical function and microstructural arrangement in Achilles tendons. It has been shown that elastic fibres present a close connection with the tenocytes. The close relationship of the three components has been revealed as a distinct, integrated and complex microstructural network. Notably, a “spiral” structure within fibril bundles in Achilles tendons was observed in some samples in specialized regions. This study substantiates the hierarchical system of the spatial microstructure of tendon, including the mapping of collagen, elastin and tenocytes, with 3‐dimensional confocal images. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1203–1214, 2017.

     

Histologic evaluation of skin-derived and collagen-based substrates implanted in palatal wounds

Tissue shortage complicates the surgery of cleft lip and palate anomalies and the healing of defects on the palate impairs growth of the dento-alveolar complex due to scar tissue formation. Implantation of substitutes into the wound area might overcome this adverse effect. The aim of this study was to compare the tissue response to three collagen-based (collagen type I substrate alone, or collagen coated with elastin or chondroitin-6-sulfate) and two skin-derived substrates (unprocessed dermis and AlloDerm) after implantation into 12 dogs. Histology was performed at 3, 10, and 20 days postsurgery. We showed that all substrates were well tolerated. However, it is unclear whether AlloDerm was rapidly degraded or if it was sequestrated. There was no elastin or collagen present in these wounds. All collagen-based substrates showed good epithelial regeneration, although heparan sulfate (JM 403) was absent. Wounds treated with the collagen-based substrates contained fewer myofibroblasts at 20 days postsurgery and the type III collagen fibers in the immature scar tissue were more randomly oriented than in an untreated wound. In conclusion, palatal wounds with a dermal substrate heal with fewer indications of scar tissue formation and evoke only a mild inflammatory reaction, which is preferred over the tissue reaction in an untreated wound.     

Fibrillin-1 and elastin are differentially expressed in hypertrophic scars and keloids.

Hypertrophic scars and keloids are two forms of excessive cutaneous scarring. Considering the importance of extracellular matrix elements in tissue repair, a morphological and quantitative analysis of the elastic system components (fibrillin-1 and elastin) was performed in normal skin, normal scars, hypertrophic scars, and keloids. In superficial and deep dermis, fibrillin-1 volume density was significantly higher in normal skin compared with normal scars, hypertrophic scars, and keloids. The fibrillin-1 volume density did not show differences between hypertrophic scars and keloids in superficial or deep dermis. In superficial dermis, elastin volume density was higher in normal skin compared with normal scars, hypertrophic scars, and keloids. In deep dermis, the elastin volume density was higher in keloids compared with normal skins, normal scars, and hypertrophic scars. We showed that the distribution of fibrillin-1 and elastin is disrupted in all kinds of scars analyzed, but there are two patterns: one for normal scars and another for excessive scars.     

DL-penicillamine induced alteration of elastic fibers of periosteum-perichondrium and associated growth inhibition: an experimental study.

Perichondrium-periosteum, being of collagen and elastic fiber, is regarded as a bone growth regulating factor. The aim of the present study was to investigate the role of collagen and elastic fibers on bone growth, by interfering with the fiber assembly in growing chicks upon administration of DL-penicillamine (DL-PNA). Our findings demonstrated that DL-PNA determined relevant modifications in the perichondrium-periosteum, as shown by histochemical, histomorphometrical,biochemical and ultrastructural analysis. This chemical has been shown to inhibit the formation of desmosine cross-links in elastin and to induce an increase of elastin associated microfibrils. On the contrary, the collagen network and the biochemical collagen markers were not affected. These changes resulted in a dramatically reduced growth of long bones in comparison with control. Perichondrial-periosteal regulation of bone growth may be mediated by mechanical and biological factors. This study demonstrates a microstructural change in the perichondrium-periosteum with decreased elastin and increased elastic microfibrils content in penicillamine treated chicks. The mechanism linking changes in the perichondrium-periosteum with altered growth still needs to be elucidated.     

大鼠异种腹主动脉移植形态学重构

目的：研究免疫排斥反应诱导移植血管损伤与重构的形态变化特征以及与腹主动脉瘤的关系。方法：选取豚鼠SD大鼠腹主动脉瘤模型．采用血管特殊染色及计算机图像半定量分析技术，分别于术后1、2、3、4周动态观测和比较移植腹主动脉平滑肌、弹力蛋白和胶原蛋白相对含量、内外径以及腔面积的变化。结果：术后移植腹主动脉各形态学指标均呈渐进性变化，其中术后1周平滑肌及胶原蛋白已明显减少，外径也明显增大；弹力蛋白于术后1周出现减少，至第2周已达最高峰；而内径以及腔面积于术后1周反而减少，3周后才明显增大。并且，弹力蛋白、平滑肌减少与腹主动脉扩张呈现显负相关。结论：异种腹主动脉移植结构重构以平滑肌反应最敏感，弹力蛋白和外径变化最显。可导致腹主动脉呈瘤样扩张。     

The effects of radiofrequency on skin: experimental study

BACKGROUND AND OBJECTIVE: Radiofrequency represents the most recent skin-rejuvenation treatment. The aim of this study was to study the changes in tissues and cells, as well as in the thickness and structure of the skin of guinea pigs following several sessions of radiofrequency. MATERIALS AND METHODS: Six sessions (1 session/week) of radiofrequency (Thermafine Lift device, Grupo Soliláser, S.L., Girona, Spain) were applied to the skin of guinea pigs. Biopsies were taken following each session and 2 months following the last application. The resultant samples were included in paraffin via the usual method, sectioned at 5 microm and stained with Haematoxylin-Eosin, Alcian blue, Toluidine blue, van Gieson's and Verhoeff's stains. These techniques were used in order to observe possible changes in the thickness and structures of the skin: mucopolysaccharides and collagen and elastic fibres. RESULTS: The most relevant changes were found in the papillary dermis which underwent an expansion due to oedema and vascular congestion (first half of the experiment), followed by an increase in cellularity and the accumulation of intercellular substance. An increase in collagen, elastic fibres and mucopolysaccharides was observed. CONCLUSION: The Thermafine Lift device caused no significant macroscopic or microscopic alterations in the skin of guinea pigs, yet increased both the thickness, as well as the mucopolysaccharide, collagen and elastic-fibre content.     

Ultrastructural investigations of the palmarfascia in M. Dupuytren (author's transl)]

The palmarfascia of 30 patients with Dupuytrens disease was investigated light- and electron microscopically. As typical changes there exist an increase in cells which form noduli. Many cells contain contractile filaments in their cytoplasma are therefore described as myofibroblasts. Beside these cells active fibroblasts could be observed. Close to their cell membranes there occur filaments with a diameter of 100--300 A. In a greater distance fibrils and fibers with a diameter of 400--600 A were observed. The regular order of the connective tissue is disturbed, one often can observe "vortex" like structures. The lumina of many capillaries are collapsed. The pericytes are increased. The nerve fibers are morphologically unchanged - they are often closely surrounded by collagen fibers. Respecting the morphological findings the pathogenesis of Dupuytrens disease is discussed.     

Ultrastructure of human normal and pathological reassembled collagen fibers]

It is possible, in vitro, to break down human skin then reassemble the collagen fibres of the dermis, essentially type I collagen. The authors have developed a biophysical process for the manufacture of type I autocollagen. They present the electron microscopic study of this collagen. These healthy collagen fibres are compared with the fibres obtained from radiation dermatitis skin and keloid scars. Major ultrastructural differences are demonstrated.     

Trichloroacetic Acid Peeling Versus Dermabrasion: A Histometric, Immunohistochemical, and Ultrastructural Comparison

Background. Trichloroacetic acid (TCA) chemical peel and dermabrasion are beneficial methods for treatment of photoaged skin.
Objective. In this study, we evaluated the changes induced by these therapies on various structures of facial skin of nine dark-skinned patients (Fitzpatrick types IV–V; TCA, five patients; dermabrasion, four patients) demonstrating different degrees of photodamage.
Methods. Routine histopathology coupled with histometric computer-assisted image analysis was used to assess epidermal changes. Alcian blue stain was used to evaluate changes in glycosaminoglycans. Immunoperoxidase techniques with antibodies against types I and III collagen and elastin were used to evaluate quantitatively changes in collagen and elastic fibers, and their ultrastructure was examined by transmission electron microscopy.
Results. Similar histologic, immunohistochemical, as well as ultrastructural changes were observed in the two groups, including epidermal and dermal rejuvenation with new collagen deposition and normalization of the elastic tissue. However, these changes were more prominent in patients treated with dermabrasion than those treated with TCA.
Conclusion. The results of this study suggest that beneficial effects of such modalities on facial skin were accomplished primarily by increasing the amounts of collagen I and collagen III and improving the morphologic appearance of collagen and elastic fibers.     

Hyaluronic acid three‐dimensional scaffold for surgical revision of retracting scars: a human experimental study

An observational study was carried out at the Plastic and Reconstructive Surgery Unit of the University of Pavia – Salvatore Maugeri Research and Care Institute, Pavia, Italy, to assess the clinical and histological long‐term outcomes of autologous skin grafting of fresh surgical wounds following previous repair with a hyaluronic acid three‐dimensional scaffold (Hyalomatrix®). Eleven fresh wounds from surgical release of retracted scars were enrolled in this study. A stable skin‐like tissue cover was observed in all of the treated wounds in an average 1 month's time; at the end of this study, after an average of 12 months' time, all of the reconstructed areas were pliable and stable, although an average retraction rate of 51·62% was showed. Histological observation and immunohistochemical analysis displayed integration of the graft within the surrounding tissues. A regenerated dermis with an extracellular matrix rich in type I collagen and elastic fibres and with reduced type III collagen rate was observed. The epidermis and dermoepidermal junction featured a normal appearance with well‐structured dermal papillae, too. Although the histological features would suggest regeneration of a skin‐like tissue, with a good dermis and no signs of scarring, the clinical problem of secondary contracture is still unsolved.     

Hautersatzverfahren in der Verbrennungschirurgie

Patients with extensive deep partial or full thickness burns require early excision of necrotic tissue, however, in many of these cases simultaneous autografting is not possible due to the general condition of the patient. In this instance temporary dressings like allogeneic or xenogeneic skin or foam dressings can be applied to minimize fluid and protein loss. In Europe glycerolized preserved allogeneic skin remains the treatment standard. Dermal replacements are considered to optimize the long-term outcome of split thickness skin grafting. Reduced contracture rates and increased pliability have been reported after additional dermal enhancement with either collagen-glycosaminoglycan matrix, acellular allogeneic dermis or collagen/elastin matrix. True regeneration of the dermis has not yet been observed. However, these materials are suitable for improvement of the wound bed and also the final result after split thickness skin transplantation.     

Structural basis for the changing physical properties of human pulmonary vessels with age

Circumferential strips of pulmonary vessel wall were obtained at necropsy from the major arterial and venous branches at the lung hilum in patients aged 7-87 years. The extensibility of these strips was measured using the tension balance method of Harris et al. (British Heart Journal, 1965, 27, 651-659). The vessels were then bisected, and half of each strip was submitted for structural analysis using morphometric methods on paraffin sections stained to show the collagen, elastin, and muscle content. The other halves of the formalin-fixed vessel strips were examined chemically to determine their collagen content by estimation of the total hydroxyproline content. The thickness of the vessel media was measured microscopically on all of the sections examined. Quantitative measurements were made on 42 arteries and 37 veins. Contrary to expectation, there was a steady fall in medial collagen content with increasing age in arteries and veins. The decrease in collagen content was similar in the morphometric and chemical studies and was statistically significant. The thickness of the vessel media did not change significantly with age. The pulmonary artery and vein strips were less extensible in the older age groups, the main change occurring in the elastic phase of the vascular stress/strain curves. It is suggested that changes in the elastic tissue at a molecular and lamellar level are responsible for the increasing stiffness of pulmonary vessels rather than changes in the medial collagen content.     

The histomorphology of the elastic tissue system in the skin of the human hand.

This study compares the effectiveness of the Verhoeff and Puchtler-Sweat resorcin fuschin methods of staining the elastic tissue of palmar and dorsal skin specimens of the human hand. The resorcin stain demonstrates the "oxytalan" fibres of the elastic tissue system in the dermal papillary layer of the palmar skin. Oxytalan fibres are not generally demonstrated by the Verhoeff method and are not demonstrated in the dorsal skin specimens. These studies substantiate the existence of a system of elastic tissue fibres in the skin of the hand. The inter-relationship of the elastic and collagenous fibres appears to complement each of their functions; namely, in anchoring the dermo-epidermal junction, absorbing the stresses of stretch and compressive forces and return the tissues to their original state of tension, and providing protection to the dermal appendages in addition to lending a gradient mobility transmitted through the dermis to the subcutaneous layer to give suppleness and mobility to the skin.     

Effects of Ethanol on Intracorporeal Structures of the Rat

Objective: Previous studies demonstrated that acute in vitro exposure of corpus cavernosal tissue to ethanol decreased its response to field stimulation and pharmacological stimulation. In the present study we investigated the effects of chronic ethanol consumption on the ultrastructure of cavernosal smooth muscle cells, elastic fibres and collagen content. Material and methods: Fourteen adult wistar rats were divided into a control group (n = 7, fed a standard diet and tap water) and an alcoholic group (n = 7, fed a standard diet and 5% (v/v) ethanol in drinking water and by increasing the ethanol concentration for every week, at the end of 6th week 30% (v/v) ethanol concentration was attained. Same dose was given until 12th week. At the end of 12th week blood samples were obtained and the ethanol concentrations were determined. The cavernosal tissues were obtained and immunohistochemical examinations were performed. Results: Immunohistochemical analysis revealed that chronic ethanol exposure markedly decreased the content of smooth muscle cells, elastic fibres and collagen type 4. Conclusion: Our findings suggest that in this animal model chronic ethanol exposure decreases the percentage of staining for smooth muscle actin, elastin, and collagen type 4 which are the key structures fundamental for erection.     

Influence of the degradation of the organic matrix on the microscopic fracture behavior of trabecular bone

In recent years, the important role of the organic matrix for the mechanical properties of bone has become increasingly apparent. It is therefore of great interest to understand the interactions between the organic and inorganic constituents of bone and learn the mechanisms by which the organic matrix contributes to the remarkable properties of this complex biomaterial. In this paper, we present a multifaceted view of the changes of bone's properties due to heat-induced degradation of the organic matrix. We compare the microscopic fracture behavior (scanning electron microscopy; SEM), the topography of the surfaces (atomic force microscopy; AFM), the condition of bone constituents [X-ray diffraction (XRD), thermogravimetric analysis (TGA), and gel electrophoresis], and the macromechanical properties of healthy bovine trabecular bone with trabecular bone that has a heat-degraded organic matrix. We show that heat treatment changes the microfracture behavior of trabecular bone. The primary failure mode of untreated trabecular bone is fibril-guided delamination, with mineralized collagen filaments bridging the gap of the microcrack. In contrast, bone that has been baked at 200 degrees C fractures nondirectionally like a brittle material, with no fibers spanning the microcracks. Finally, bone that has been boiled for 2 h in PBS solution fractures by delamination with many small filaments spanning the microcracks, so that the edges of the microcracks become difficult to distinguish. Of the methods we used, baking most effectively weakens the mechanical strength of bone, creating the most brittle material. Boiled bone is stronger than baked bone, but weaker than untreated bone. Boiled bone is more elastic than untreated bone, which is in turn more elastic than baked bone. These studies clearly emphasize the importance of the organic matrix in affecting the fracture mechanics of bone.     

真皮多能干细胞移植对大鼠光老化皮肤组织的修复作用

目的:进一步探讨真皮多能干细胞(dermal multipotential stem cells,DMSCs)移植对大鼠皮肤光老化(skin photoage)后组织修复作用的影响。方法:SD大鼠58只随机分为正常对照组(8只)、细胞移植组(25只)和PBS对照组(25只),细胞移植组和模型对照组采用UVA和UVB紫外灯同时进行照射制备皮肤光老化模型。体外分离培养DMSCs,吸取1ml细胞悬液(约1×106个)或等量PBS液分别于皮肤光老化区真皮层内多点注射移植,正常对照组不做任何处理。移植后4周、8周分别采用VG染色和Weigert染色观察皮肤全层胶原纤维、弹力纤维的表达变化;同时应用免疫组织化学染色观察各组大鼠皮肤真皮层vimentin的表达变化。结果:皮肤光老化损伤后,与对照组相比,DSMCs治疗组大鼠受损皮肤组织出现较多的新生胶原纤维和弹力纤维;DMSCs移植治疗组大鼠在真皮层内vimentin染色阳性的成纤维细胞的数量明显多于PBS对照组,移植后4周主要分布于真皮浅层,8周后广泛均匀弥散在皮肤各层。结论:DMSCs移植对大鼠光老化皮肤具有明显的促进组织修复的作用。     

Development of a Tissue‐Engineered Artificial Ligament: Reconstruction of Injured Rabbit Medial Collateral Ligament With Elastin‐Collagen and Ligament Cell Composite Artificial Ligament

Ligament reconstruction using a tissue‐engineered artificial ligament (TEAL) requires regeneration of the ligament‐bone junction such that fixation devices such as screws and end buttons do not have to be used. The objective of this study was to develop a TEAL consisting of elastin‐coated polydioxanone (PDS) sutures covered with elastin and collagen fibers preseeded with ligament cells. In a pilot study, a ring‐type PDS suture with a 2.5 mm (width) bone insertion was constructed with/without elastin coating (Ela‐coat and Non‐coat) and implanted into two bone tunnels, diameter 2.4 mm, in the rabbit tibia (6 cases each) to access the effect of elastin on the bond strength. PDS specimens taken together with the tibia at 6 weeks after implantation indicated growth of bone‐like hard tissues around bone tunnels accompanied with narrowing of the tunnels in the Ela‐coat group and not in the Non‐coat group. The drawout load of the Ela‐coat group was significantly higher (28.0 ± 15.1 N, n = 4) than that of the Non‐coat group (7.6 ± 4.6 N, n = 5). These data can improve the mechanical bulk property of TEAL through extracellular matrix formation. To achieve this TEAL model, 4.5 × 10⁶ ligament cells were seeded on elastin and collagen fibers (2.5 cm × 2.5 cm × 80 µm) prior to coil formation around the elastin‐coated PDS core sutures having ball‐shape ends with a diameter of 2.5 mm. Cell‐seeded and cell‐free TEALs were implanted across the femur and the tibia through bone tunnels with a diameter of 2.4 mm (6 cases each). There was no incidence of TEAL being pulled in 6 weeks. Regardless of the remarkable degradation of PDS observed in the cell‐seeded group, both the elastic modulus and breaking load of the cell‐seeded group (n = 3) were comparable to those of the sham‐operation group (n = 8) (elastic modulus: 15.4 ± 1.3 MPa and 18.5 ± 5.7 MPa; breaking load: 73.0 ± 23.4 N and 104.8 ± 21.8 N, respectively) and higher than those of the cell‐free group (n = 5) (elastic modulus: 5.7 ± 3.6 MPa; breaking load: 48.1 ± 11.3 N) accompanied with narrowed bone tunnels and cartilage matrix formation. These data suggest that elastin increased the bond strength of TEAL and bone. Furthermore, our newly developed TEAL from elastin, collagen, and ligament cells maintained the strength of the TEAL even if PDS was degraded.     

Histologic and Ultrastructural Evaluation of Sutures Used for Surgical Fixation of the SMAS

In extensive SMAS face-lift surgery, retaining ligaments are released, and the SMAS is resutured to the deep fascia to maintain the advanced position. The suture used to reattach the SMAS should replicate the quality of support provided by the original ligaments. Nonabsorbable sutures (monofilament and braided) retrieved intraoperatively from 22 patients undergoing secondary face-lift procedures were examined by light microscopy and transmission electronmicroscopy. A distinctive enclosure of dense collagen and elastin formed around both types of suture. Based on the presence of inflammatory cells, fibroblasts, collagen, and elastin, the tissue reaction to monofilament suture was less than with the braided suture. The collagen and elastin were thicker around the braided suture, and, additionally the collagen matrix infiltrated between the individual filaments. Ultrastructural analysis of the braided suture showed significant collagen binding around each individual filament. The greater quantity of connective tissue around the thread which continued into the interstices of the braided suture has the characteristics of a ligament. This suggests a stronger and more lasting tissue fixation.     

Percutaneous collagen induction therapy: An alternative treatment for burn scars

Objective

This study aims to evaluate percutaneous collagen induction (PCI) in post-burn scarring.

Background

Patients with scarring after burn frequently request help in improving the aesthetic appearance of their residual cicatricial deformity. Their scars are generally treated by tissue transfer, W- and Z-plasties, flaps, cortisone injections or ablative procedures that injure or destroy the epidermis and its basement membrane and subsequently lead to fibrosis of the papillary dermis. The ideal treatment would be to preserve the epidermis and promote normal collagen and elastin formation in the dermis.

Patients

A total of 16 consecutive patients (average age: 37 ± 15.5 years, average body mass index (BMI): 25.7) in Germany with post-burn scarring.

Intervention

PCI using the Medical Roll-CIT (Vivida, Cape Town, South Africa). This device was designed to multiply-puncture the skin to the level of the dermal scar to institute remodelling. Patients were prepared with topical vitamin A and C cosmetic creams for a minimum of 4 weeks preoperatively to maximise collagen stimulation.

Outcome measures

The outcome was measured rating (visual analogue scale (VAS) and Vancouver Scar Scale (VSS)), histological specimen 12 months after intervention.

Results

On average, patients rated their improvement as a mean of 80% better (±15.5) than before treatment. Histologic examination revealed considerable increase in collagen and elastin deposition 12 months postoperatively. The epidermis demonstrated 45% thickening of stratum spinosum and normal rete ridges as well as the normalisation of the collagen/elastin matrix in the reticular dermis at 1 year postoperatively.

Conclusions

This pilot study shows that PCI appears to be a safe method for treating post-burn scarring without destroying the epidermis. The procedure can be repeated safely and is also applicable in regions where laser treatments and deep peels are of limited use. However, it is necessary to initiate an efficacy trial to prove the data of this pilot study.     

Abnormal deposition of collagen/elastic vascular fibres and prognostic significance in idiopathic interstitial pneumonias

BACKGROUND: Vascular remodelling has recently been shown to be a promising pathogenetic indicator in idiopathic interstitial pneumonias (IIPs). AIM: To validate the importance of the collagen/elastic system in vascular remodelling and to study the relationships between the collagen/elastic system, survival and the major histological patterns of IIPs. METHODS: Collagen/elastic system fibres were studied in 25 patients with acute interstitial pneumonia/diffuse alveolar damage, 22 with non-specific interstitial pneumonia/non-specific interstitial pneumonia and 55 with idiopathic pulmonary fibrosis/usual interstitial pneumonia. The Picrosirius polarisation method and Weigert's resorcin-fuchsin histochemistry and morphometric analysis were used to evaluate the amount of vascular collagen/elastic system fibres and their association with the histological pattern of IIPs. The association between vascular remodelling and the degree of parenchymal fibrosis in usual interstitial pneumonia (UIP) was also considered. RESULTS: The vascular measurement of collagen/elastic fibres was significantly higher in UIP than in the lungs of controls, and in those with diffuse alveolar damage and those with non-specific interstitial pneumonia. In addition, the increment of collagen/elastic fibres in UIP varied according to the degree and activity of the parenchymal fibrosis. The most important predictors of survival in UIP were vascular remodelling classification and vascular collagen deposition. CONCLUSION: A progressive vascular fibroelastosis occurs in IIP histological patterns, probably indicating evolutionarily adapted responses to parenchymal injury. The vascular remodelling classification and the increase in vascular collagen were related to survival in IIP and possibly play a role in its pathogenesis. Further studies are needed to determine whether this relationship is causal or consequential.