Tension decrease during skin stretching in undermined versus not undermined skin: an experimental study in piglets

In a controlled study using 15 piglets, the efficacy of skin stretching using a skin stretching device was tested by quantifying the tension decrease during skin stretching in undermined and not undermined wounds. The viability of the skin margins was examined in both situations. Thirty standardized wounds was created: around 15 wounds on one flank, the surrounding skin was undermined; whereas around the 15 wounds on the opposite flank, the surrounding skin was not undermined. The force required to close the 9 x 9 cm defect was measured at the beginning, after undermining, and after 30 minutes of skin stretching. Also examined was the wound healing after 1 day and 1 week. A tension decrease of 3.02 N (13.6 percent reduction of the total force that is required to close the wound at the beginning) was seen due to undermining the surrounding skin. Skin stretching for 30 minutes without undermining the skin showed a tension decrease of 6.10 N (26.5 percent). Therefore, the tension decrease due to skin stretching was twice as high in comparison with undermining the skin margins alone. This has been statistically proven to be significant (-d (difference) = 3.08, 95 percent confidence interval = 2.16; 4.00, p < 0.001). When the undermined skin of the wound was stretched for 30 minutes, we measured a total tension decrease of 7.60 N (34.1 percent). There was a statistically significant but small difference in total tension decrease as a result of undermining combined with skin stretching in comparison with skin stretching without undermining (-d = 1.51, 95 percent confidence interval = 0.77; 2.23, p < 0.001). Undermining the surrounding skin involved cutting musculocutaneous perforating vessels. Looking at the viability of the skin, seven wounds, all found in the undermined group, showed skin necrosis after 1 week. Excessive seroma formation was seen in all wounds around which the skin was undermined. In the not undermined wounds, there were no problems in wound healing. In conclusion, skin stretching for only 30 minutes using a skin stretching device significantly reduces wound closing tension. The additional advantage of skin stretching over that of undermining alone is clearly shown. Undermining the wound margins before skin stretching gives a small additional tension decrease but has well-known complications, such as skin-edge necrosis and seroma formation.     

Oxygenation and microcirculation during skin stretching in undermined and nonundermined skin

The aim of this experimental study was to assess the skin microcirculation of undermined and nonundermined wound edges closed with a skin-stretching device. In eight piglets, 9 x 9-cm wounds were created on both flanks by excision of the skin and the subcutaneous layer down to the muscular fascia, with general anesthesia. On one flank, the surrounding skin was completely undermined. For a period of 30 minutes, wound closure was performed with a stretching device, using the principle of load cycling. The device stretched the skin and moved the opposing wound edges toward each other. During this period, laser Doppler flowmetry and transcutaneous oximetry were simultaneously used to monitor microcirculation and oxygenation in the stretched skin of both flanks. Undermining of the surrounding skin produced a 12 percent decrease in the laser Doppler flowmetry signal and a 21 percent decrease in the transcutaneous oximetry value. Skin stretching resulted in decreases in the laser Doppler flowmetry signals and the transcutaneous oximetry values, whether or not the skin was undermined. Releasing the stretching device resulted in rapid normalization of the laser Doppler flowmetry values in undermined and nonundermined skin and a slow return of the transcutaneous oximetry values to close to baseline levels in nonundermined skin. The transcutaneous oximetry values in undermined skin did not return to baseline levels; each period of skin stretching resulted in an additional decrease in the transcutaneous oximetry values. Stretching of undermined skin for 30 minutes produced a significant (p < 0.0001) decrease in skin oxygenation. As a result, 50 percent of the undermined stretched skin demonstrated skin necrosis at the wound edges, which was still present after 1 week. Wound healing in the nonundermined stretched skin proceeded without problems. It is concluded from these experiments that the viability of undermined skin becomes compromised as a result of significantly decreased oxygen availability in the skin during and after stretching. Consequently, it is recommended that skin stretching be performed on nonundermined skin, rather than undermined skin. In addition, when skin is stretched to close a large defect, it is logical to use cyclic loading, so that recuperation of the skin circulation can occur. Furthermore, laser Doppler flowmetry seemed to produce atypical signals in monitoring of skin viability of wound edges closed with a skin-stretching device.     

A quantitative method to determine the orientation of collagen fibers in the dermis.

We have developed a quantitative microscopic method to determine changes in the orientation of collagen fibers in the dermis resulting from mechanical stress. The method is based on the use of picrosirius red-stained cryostat sections of piglet skin in which collagen fibers reflect light strongly when epipolarization microscopy is used. Digital images of sections were converted into binary images that were analyzed quantitatively on the basis of the length of the collagen fibers in the plane of the section as a measure for the orientation of the fibers. The length of the fibers was expressed in pixels and the mean length of the 10 longest fibers in the image was taken as the parameter for the orientation of the fibers. To test the procedure in an experimental setting, we used skin after 0 and 30 min of skin stretching. The orientation of the fibers in sections of control skin differed significantly from the orientation of fibers in sections of skin that was stretched mechanically for 30 min [76 +/- 15 (n=5) vs 132 +/- 36 (n=5)]. The method described here is a relatively simple way to determine (changes in) the orientation of individual collagen fibers in connective tissue and can also be applied for analysis of the orientation of any other structural element in tissues so long as a representative binary image can be created.     

Vanadate ingestion increases the gain in wound breaking strength and leads to better organized collagen fibers in rats during healing

Repair of incision wounds closed by suturing is evaluated by the progressive gain in wound breaking strength. Previously the closure of open wounds in rats ingesting vanadate, an inhibitor of tyrosine phosphate phosphatases, was shown to occur with deposition of more uniformly organized collagen fiber bundles. The hypothesis of this study was that deposition of more uniformly organized collagen fibers would enhance the gain in wound breaking strength of incisional wounds. Six adult rats received vanadate-supplemented saline drinking water for 1 week before placement of two 6-cm, parallel, suture-closed wounds on their backs. Six control rats received identical wounds and were given saline drinking water. The drinking water regimen was continued for 1 week after wounding, and then wound strength was tested with a tensiometer and tissue samples were obtained for histologic evaluation. Wound breaking strength doubled in vanadate-treated rats compared with controls. Bright-field and polarized light microscopy showed that the connective tissue matrix of granulation tissue from control rats was oriented perpendicular to the surface of the skin. In contrast, the connective tissue matrix of granulation tissue from vanadate-treated rats was oriented parallel to the skin surface. The gap in granulation tissue between the edges of the wounds in the vanadate-treated rats was greater than that in controls. Electron microscopy showed that wounds in the vanadate-treated contained uniform collagen fibers that were 20 percent greater in diameter and more evenly spaced than they were in controls. It is proposed that these changes in the organization of collagen fibers within incisional wounds were responsible for the increased wound breaking strength observed in rats ingesting vanadate.     

Relationship between mechanical properties and collagen structure of closed and open wounds

Mechanical properties and collagen structure of excisional wounds left open are compared with wounds closed by clips. In both wound models, collagen fiber diameter increases with time post-wounding and is related to tensile strength. Clipped wounds show a higher ultimate tensile strength and tangent modulus compared with open wounds. In clipped wounds, newly deposited collagen appears as a biaxially oriented network as observed in normal skin. In open wounds a delay in the organization of the collagen network is observed and parallel wavy-shaped ribbons of collagen fibers are deposited. At long term, the high extensibility observed in open wounds may be due to the sliding of ribbons of collagen fibers past each other.     

Induction of tenascin in healing wounds

The distribution of the extracellular matrix glycoprotein, tenascin, in normal skin and healing skin wounds in rats, has been investigated by immunohistochemistry. In normal skin, tenascin was sparsely distributed, predominantly in association with basement membranes. In wounds, there was a marked increase in the expression of tenascin at the wound edge in all levels of the skin. There was also particularly strong tenascin staining at the dermal-epidermal junction beneath migrating, proliferating epidermis. Tenascin was present throughout the matrix of the granulation tissue, which filled full-thickness wounds, but was not detectable in the scar after wound contraction was complete. The distribution of tenascin was spatially and temporally different from that of fibronectin, and tenascin appeared before laminin beneath migrating epidermis. Tenascin was not entirely codistributed with myofibroblasts, the contractile wound fibroblasts. In EM studies of wounds, tenascin was localized in the basal lamina at the dermal-epidermal junction, as well as in the extracellular matrix of the adjacent dermal stroma, where it was either distributed homogeneously or bound to the surface of collagen fibers. In cultured skin explants, in which epidermis migrated over the cut edge of the dermis, tenascin, but not fibronectin, appeared in the dermis underlying the migrating epithelium. This demonstrates that migrating, proliferating epidermis induces the production of tenascin. The results presented here suggest that tenascin is important in wound healing and is subject to quite different regulatory mechanisms than is fibronectin.     

THE DYNAMICS OF WOUND CLOSURE AND ITS ROLE IN THE PROGRAMMING OF PLANARIAN REGENERATION. II — DISTALIZATION

When the dorsal and ventral epidermal layers join by first intention during the closure of the wound, the cells of their borders (M-cells) do not meet in the same manner in all sections. In anterior sections the dorsal M-cells attach themselves to the ventral basement membrane, so that only the dorsal epidermis is stretched. In posterior sections the dorsal and the ventral M-cells join by their apical edges without being closely apposed to the wound surface. Only the ventral cells are stretched because of their specific motility. In longitudinal sections the dorsal and the ventral M-cells also join by their apical edges, but since they are closely apposed to the wound surface both epidermal layers are stretched. The stretching is a process equivalent to distalization. The junction between the dorsal and the ventral epidermis is shifted ventrally in the anterior wounds (as in the intact heads) and dorsally in the posterior wounds (as in the intact tails). Some abnormalities of wound closure have been observed at levels where heteromorphic regeneration frequently occurs. These findings are consistent with the hypothesis previously advanced (3) that the modalities of wound closure establish the programme for regeneration.     

Fibronectin involvement in granulation tissue and wound healing in rabbits

This study describes the distribution of fibronectin and its association with reticulin fibers (type III collagen) and hyaluronic acid in shallow rabbit wounds. Linear incisions were made dorsally with a surgical blade. Animals were sacrificed and 1,2,3,4,5, and 8 day wounds were examined using peroxidase-antiperoxidase to localize affinity-purified antibodies to fibronectin. Tissue samples were also stained with hematoxylin and eosin in addition to silver stains for reticulin, and Alcian blue for hyaluronic acid. After wounding, the incision filled with a fibrin clot that stained positively for fibronectin. The underlying dermis and adjacent, unwounded dermis also contained fibronectin. Epidermal cells that migrate from the wound margin between the clot and the dermis were in direct association with fibronectin in these wound components. By 72 hr, epidermal continuity was reestablished. Early granulation tissue formation was apparent just below the epidermis 5 day wounds. Fibronectin was observed in the matrix surrounding individual fibroblasts and codistributed with reticulin fibers and hyaluronic acid in both 5 and 8 day wounds. Granulation tissue of 8 day wounds stained intensely for fibronectin and extended to a greater depth in the reticular dermis. Dense fibrillar networks of fibronectin and fibroblasts were aligned parallel to the epidermis, giving the granulation tissue a highly structured and organized appearance. Fibroblasts contained fibronectin and were surrounded by less fibronectin at the wound periphery than within the granulation tissue. These findings suggest that fibronectin may be important in the reconstruction of tissues during repair by functioning as an extracellular scaffold for migrating cells.     

Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition.

Fetal wounds pass from scarless repair to healing with scar formation during gestation. This transition depends on both the size of the wound and the gestational age of the fetus. This study defines the transition period in the fetal rat model and provides new insight into scarless collagen wound architecture by using confocal microscopy. A total of 16 pregnant Sprague-Dawley rats were operated on. Open full-thickness wounds, 2 mm in diameter, were created on fetal rats at gestational ages 14.5 days (E14; n = 10), 16.5 days (E16; n = 42), and 18.5 days (E18; n = 42) (term = 21.5 days). Wounds were harvested at 24 (n = 18 per gestational age) and 72 hours (n = 24 per gestational age). Skin at identical gestational ages to wound harvest was used for controls. The wounds were fixed and stained with hematoxylin and eosin, antibody to type I collagen, and Sirius red for confocal microscopic evaluation. No E14 rat fetuses survived to wound harvest. Wounds created on E16 fetal rats healed completely and without scarring. E16 fetal rat hair follicle formation and collagen architecture was similar to that of normal, nonwounded skin. Wounds created on E18 fetal rats demonstrated slower healing; only 50 percent were completely healed at 72 hours compared with 100 percent of the E16 fetal rat wounds at 72 hours. Furthermore, the E18 wounds healed with collagen scar formation and without hair follicle formation. Confocal microscopy demonstrated that the collagen fibers were thin and arranged in a wispy pattern in E16 fetal rat wounds and in nonwounded dermis. E18 fetal rat wounds had thickened collagen fibers with large interfiber distances. Two-millimeter excisional E16 fetal rat wounds heal without scar formation and with regeneration of normal dermal and epidermal appendage architecture. E18 fetal rat wounds heal in a pattern similar to that of adult cutaneous wounds, with scar formation and absence of epidermal appendages. Confocal microscopy more clearly defined the dermal architecture in normal skin, scarless wounds, and scars. These data further define the transition period in the fetal rat wound model, which promises to be an effective system for the study of in vivo scarless wound healing.     

Effect of fibroblast implants on wound healing of irradiated skin: assay of wound strength and quantitative immunohistology of collagen

The role of dermal fibroblasts in the expression of radiation-induced damage to the skin was studied. Fibroblasts from neonatal mice were cultured, harvested, and injected into full-depth surgical incisions in the dorsal area of mouse skin, which had been previously locally irradiated by 18 Gy X rays. As a control, cells irradiated with a dose of 20 Gy were also injected. The effect of radiation and fibroblast implants on the gain of skin wound strength was assayed. In an additional experiment freshly isolated cells were implanted. Two weeks following wounding the irradiated skin had reached only about a third of the strength of unirradiated skin. A significant increase of wound strength in irradiated skin was observed when 1.5-2 x 10(6) cultured fibroblasts or freshly isolated fibroblasts were injected into the 20-mm-long wound bed. Irradiated cells had significantly less effect. This suggests that implanting isolated syngeneic cells may "rescue" wounds from the effect of prior irradiation. Semiquantitative immunohistology of types I and III collagen was performed in parallel using a video image digitizing system. Levels of both types I and III collagen were altered in the dermis and the wound tissues in irradiated skin, but the implant of cultured fibroblasts did not affect notably the total levels and the disposition of the two collagen isotypes.     

Identification of the depth of burn injury by collagen stainability

Heat-denatured collagen in burned skin stains red instead of blue in Masson's trichrome stain. This change in stainability corresponds to the loss of birefringence in slides examined in polarized light. The depth of the abnormal staining of the skin slices was proportional to the time and temperature of the heat exposure. It is concluded that the change in collagen stainability from blue to red relates to the loss of crystallinity or parallel alignment of the collagen fibers. It is further proposed that change in the stainability of collagen in the burns could be used to delineate the depth of the thermal skin injury or the effectiveness of the surgical excision or debridement of the wound by dressing materials.     

Mice that lack matrix metalloproteinase-9 display delayed wound healing associated with delayed reepithelization and disordered collagen fibrillogenesis

Matrix metalloproteinase- (MMP-9) is involved in processes that occur during cutaneous wound healing such as inflammation, matrix remodeling, and epithelialization, To investigate its role in healing, full thickness skin wounds were made in the dorsal region of MMP-9-null and control mice and harvested up to 14 days post wounding. Gross examination and histological and immunohistochemical analysis indicated delayed healing in MMP-9-null mice. Specifically, MMP-9-null wounds displayed compromised reepithelialization and reduced clearance of fibrin clots. In addition, they exhibited abnormal matrix deposition, as evidenced by the irregular alignment of immature collagen fibers. Despite the presence of matrix abnormalities, MMP-9-null wounds displayed normal tensile strength. Ultrastructural analysis of wounds revealed the presence of large collagen fibrils, some with irregular shape. Keratinocyte proliferation, inflammation, and angiogenesis were found to be normal in MMP-9-null wounds. In addition, VEGF levels were similar in control and MMP-9-null wound extracts. To investigate the importance of MMP-9 in wound reepithelialization we tested human and murine keratinocytes in a wound migration assay and found that antibody-based blockade of MMP-9 function or MMP-9 deficiency retarded migration. Collectively, our observations reveal defective healing in MMP-9-null mice and suggest that MMP-9 is required for normal progression of wound closure.     

Effect of topically applied steroidal and nonsteroidal anti-inflammatory agents on skin repair and regeneration

We studied the effects of topically applied steroidal and nonsteroidal anti-inflammatory agents on dermal and epidermal wound healing. Superficial wounds (0.3 mm deep) on the skin of domestic pigs were treated daily with either 0.1% triamcinolone acetonide (TA), 1% hydrocortisone (HC), 1% nandrolone decanoate (ND), 1% ND + 0.1% TA, 10 mg ibuprofen, 10 mg meclofenamate sodium, 3 mg indomethacin, vehicle (USP petrolatum or 70% ethanol), or control (untreated). Wounds were excised on days 2-7 after wounding and the epidermis was separated from the dermis. The dermis was assayed for collagen biosynthesis and the epidermis was evaluated for reepithelialization. A significant decrease (P less than 0.01) in relative collagen synthesis was observed in the wounded dermis in both HC- and TA-treated groups on day 3 after wounding, but there were no significant differences on days 4-7. Depressed collagen and noncollagenous protein production was also noted in vehicle-treated wounds on day 3. Topical application of ND did not affect collagen synthesis, but when combined with TA it eliminated the inhibitory effect observed as a result of TA alone. Topical ND accelerated wound reepithelialization by 12.5% compared with vehicle and by 26% compared with untreated controls. TA delayed epidermal resurfacing by 22%, but when combined with ND (ND + TA) the rate of reepithelialization was similar to vehicle-treated wounds. HC enhanced resurfacing when compared with untreated wounds but did not differ markedly from its vehicle. The nonsteroidal anti-inflammatory drugs when topically applied markedly reduced inflammation (erythema, heat, and edema) but did not influence the healing process.     

SPARC-null mice exhibit accelerated cutaneous wound closure.

Expression of SPARC (secreted protein acidic and rich in cysteine; osteonectin, BM-40), an extracellular matrix (ECM) associated protein, is coincident with matrix remodeling. To further identify the functions of SPARC in vivo, we have made excisional wounds on the dorsa of SPARC-null and wild-type mice and monitored closure over time. A significant decrease in the size of the SPARC-null wounds, in comparison to that of wild-type, was observed at Day 4 and was maximal at Day 7. Although substantial differences in the percentage of proliferating cells were not apparent in SPARC-null relative to wild-type wounds, primary cultures of SPARC-null dermal fibroblasts displayed accelerated migration, relative to wild-type fibroblasts, in wound assays in vitro. Although the expression of collagen I mRNA in wounds, as measured by in situ hybridization (ISH), was not significantly different in SPARC-null vs wild-type mice, the collagen content of unwounded skin appeared to be substantially lower in the SPARC-null animals. By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of wild-type skin. Moreover, we found an inverse correlation between the efficiency of collagen gel contraction by dermal fibroblasts and the concentration of collagen within the gel itself. We propose that the accelerated wound closure seen in SPARC-null dermis results from its decreased collagen content, a condition contributing to enhanced contractibility.     

The effect of stretching on formation of myofibroblasts in mouse skin

Summary Wound contraction results from the contractile activity of modified fibroblasts, termed myofibroblasts, which are present in the granulation tissue of the healing wound. This study examines the relative role of mechanical tension (stretching) and wound healing as events capable of stimulating the formation of myofibroblasts in mouse skin. The skin of hairless mice was subjected to mechanical stretching and to a small incisional wound either separately or in combination. Animals were killed at intervals between 1 and 6 days and the dermis examined with the electron microscope. Stretching alone produced little evidence of inflammation at any time interval but cells with the ultrastructural characteristics of myofibroblasts were present at 4 days and abundant at 6 days. Skin that had been both stretched and wounded showed a marked inflammatory response and also contained myofibroblasts, but they were less frequent than in the skin subjected to stretching alone. Very few myofibroblasts were evident in skin that had only been wounded. It is suggested that the effect of mechanical tension alone may initiate formation of myofibroblasts in a tissue.     

Arrangement of microfibrils in collagen fibrils of tendons in the rat tail

Summary The microfibrillar arrangement in collagen fibrils of tendons in the tail of the rat was examined by electron microscopy and X-ray diffraction. Fresh and air-dried collagen fibers were examined in unstretched and stretched conditions. The results demonstrate that the microfibrils have a course parallel to the longitudinal axis of the collagen fibrils. The influence of stretching and hydration of the samples on the orientation of fibrils and microfibrils is also assessed.     

Local distribution of collagen fibers determines crack initiation site and its propagation direction during aortic rupture

Although elucidation of the mechanism of aortic aneurysm rupture is important, the characteristics of crack initiation and propagation sites remain unknown. To determine the microscopic properties of these sites, the characteristics of local strains and constituents at crack initiation and propagation sites were investigated during biaxial stretching of porcine thoracic aortas (PTAs). PTAs were sliced into approximately 50-\(\upmu \hbox {m}\)-thick sections, and the center of the sections was made especially thin using our previously developed technique. Alpha-elastin and cell nuclei were fluorescently labeled as indices of local elastin density and as a strain marker, respectively. Birefringence and second harmonic generation (SHG) light images were used to determine local collagen distributions. The specimens were then stretched biaxially with a laboratory-made tensile tester under a fluorescent microscope equipped with a birefringence imaging system. Local strains were calculated from the local displacement of the cell nuclei. The degree of alignment and density of local collagen fibers were measured from retardance and SHG images. The strain distributions, specifically the first and second principal, and maximum shear strains, fluorescent intensity of \(\upalpha \)-elastin, and degree of alignment of collagen fibers, showed insignificant differences between the crack initiation sites and other sites. The retardance and intensity of SHG light at the crack initiation sites were significantly lower than those at other sites for all (\(n = 6\)) specimens. Cracks tended to propagate along the local direction of the collagen fibers. These results indicate that the local density and direction of collagen fibers play an important role in aorta rupture.     

Striae albae: a morphological study on the human skin

Specimens of abdomen skin, comprising alternate areas of striae albae and healthy skin, were removed during surgical lipectomy from multiparous and obese women between the ages of 24 and 53 years. A flattening and thinning of the striae albae surface and the almost complete disappearance of dermal papillae was observed in paraffin and thin sections. The papillary dermis was found to be almost completely replaced by straight bundles of collagen fibres running parallel to the skin surface. Immunofluorescence data revealed in these bundles high positivity for type I collagen. The underlying reticular dermis was also found to contain large densely packed bundles of collagen fibres running parallel to the skin surface. Both papillary and reticular dermis collagen fibres were mainly arranged orthogonally to the main axis of the stria. Furthermore, the density of the collagen fibre bundles and the diameter of the collagen fibrils was found to be greater than that of the clinically healthy skin. A larger number of elastic fibres, which presented an abnormal ultrastructural appearance, were visible in pathological papillary and reticular dermis.     

Monitoring dynamic collagen reorganization during skin stretching with fast polarization‐resolved second harmonic generation imaging

The mechanical properties of biological tissues are strongly correlated to the specific distribution of their collagen fibers. Monitoring the dynamic reorganization of the collagen network during mechanical stretching is however a technical challenge, because it requires mapping orientation of collagen fibers in a thick and deforming sample. In this work, a fast polarization‐resolved second harmonic generation microscope is implemented to map collagen orientation during mechanical assays. This system is based on line‐to‐line switching of polarization using an electro‐optical modulator and works in epi‐detection geometry. After proper calibration, it successfully highlights the collagen dynamic alignment along the traction direction in ex vivo murine skin dermis. This microstructure reorganization is quantified by the entropy of the collagen orientation distribution as a function of the stretch ratio. It exhibits a linear behavior, whose slope is measured with a good accuracy. This approach can be generalized to probe a variety of dynamic processes in thick tissues.      

Ex-vivo observation of calcification process in chick tibia slice: Augmented calcification along collagen fiber orientation in specimens subjected to static stretch

Bone formation through matrix synthesis and calcification in response to mechanical loading is an essential process of the maturation in immature animals, although how mechanical loading applied to the tissue increases the calcification and improves mechanical properties, and which directions the calcification progresses within the tissue are largely unknown. To address these issues, we investigated the calcification of immature chick bone under static tensile stretch using a newly developed real-time observation bioreactor system. Bone slices perpendicular to the longitudinal axis obtained from the tibia in 2- to 4-day-old chick legs were cultured in the system mounted on a microscope, and their calcification was observed up to 24 h while they were stretched in the direction parallel to the slice. Increase in the calcified area, traveling distance and the direction of the calcification and collagen fiber orientation in the newly calcified region were analyzed. There was a significant increase in calcified area in the bone explant subjected to tensile strain over ∼3%, which corresponds to the threshold strain for collagen fibers showing alignment in the direction of stretch, indicating that the fiber alignment may enhance tissue calcification. The calcification progressed to a greater distance to the stretching direction in the presence of the loading. Moreover, collagen fiber orientation in the calcified area in the loaded samples was coincided with the progression angle of the calcification. These results clearly show that the application of static tensile strain enhanced tissue calcification, which progresses along collagen fibers aligned to the loading direction.