Investigations of Human Fascia Lata Elemental Composition-the Effect of Different Preservation and Mineralisation Methods.

Influence of fixation medium and storage conditions as well as impact of sample mineralisation procedure on determination of minerals in human fascia lata was examined and discussed. Freezing and storage in 10% neutral buffered formalin solution and in 2.5% glutaraldehyde were used as the preservation methods of the samples. The concentrations of, both essential and toxic, elements were measured by ICP-OES method in fascia lata samples mineralised with concentrated nitric acid in a closed microwave system and in open vessels heated on a hot plate. Freezing was found as the best preserving method of fascia lata samples because of the number of elements determined and determination precision. The trace element (Cd, Cr, Cu, Fe, Ni, Sr, Zn) concentrations obtained in samples decomposed using the conventional hot plate were different from analogous measurements in solutions obtained after application of closed vessels and microwave energy assistance. Differences between the mineral compositions of fascia samples variously preserved and mineralised were statistically evaluated and discussed. Interelement correlations were analysed taking into account an impact of various methods of sample conservation. Strong, positive association between element content was discovered for Cr-Ba, Mn-Ba, P-Ba, Sr-Ba, Sr-Ca, Zn-Ca, Mn-Cr, Pb-Cr, Sr-Cr, Mg-Fe, P-Fe, Pb-Ni, Ti-Ni and Sr-P pairs of elements.

Enhanced vascular biocompatibility of decellularized xeno-/allogeneic matrices in a rodent model.

Glutaraldehyde preservation is the gold standard for cardiovascular biological prosthesis. However, secondary calcifications and the absence of tissue growth remain major limitations. Our study assessed in vitro and in vivo the biocompatibility of human (fascia lata, pericardium) and porcine tissues (pericardium, peritoneum) treated with a physicochemical procedure for decellularization and non-conventional pathogens inactivation. Biopsies were performed before and after treatment to assess decellularization (HE/Dapi staining/DNA quantification/MHC I/alpha gal immunostaining) and mechanical integrity. Forty-five rats received an abdominal aortic patch of native cryopreserved tissues (n = 20), treated tissues (n = 20) or glutaraldehyde-preserved bovine pericardium (GBP, control, n = 5). Grafts were explanted at 4 weeks and processed for HE/von Kossa staining and immunohistochemistries for lymphocytes (CD3)/macrophages (CD68) histomorphometry. 95% of decellularization was obtained for all tissues except for fascia lata (75%). Mechanical properties were slightly altered. In the in vivo model, a significant increase of CD3 and CD68 infiltrations was found in native and control implants in comparison with decellularized tissues (p < 0.05). Calcifications were found in 3 controls. Decellularized tissues were recolonized. GBP showed the most inflammatory response. This physicochemical treatment improves the biocompatibility of selected xeno/allogeneic tissues in comparison with their respective native cryopreserved tissues and with GBP. Incomplete decellularization is associated with a significantly higher inflammatory response. Our treatment is a promising tool in the field of tissue decellularization and tissue banking.

Directional differences in the biaxial material properties of fascia lata and the implications for fascia function.

Fascia is a highly organized collagenous tissue that is ubiquitous in the body, but whose function is not well understood. Because fascia has a sheet-like structure attaching to muscles and bones at multiple sites, it is exposed to different states of multi- or biaxial strain. In order to measure how biaxial strain affects fascia material behavior, planar biaxial tests with strain control were performed on longitudinal and transversely oriented samples of goat fascia lata (FL). Cruciform samples were cycled to multiple strain levels while the perpendicular direction was held at a constant strain. Structural differences among FL layers were examined using histology and SEM. Results show that FL stiffness, hysteresis, and strain energy density are greater in the longitudinal vs. transverse direction. Increased stiffness in the longitudinal layer is likely due to its greater thickness and greater average fibril diameter compared to the transverse layer(s). Perpendicular strain did not affect FL material behavior. Differential loading in the longitudinal vs. transverse directions may lead to structural changes, enhancing the ability of the longitudinal FL to transmit force, store energy, or stabilize the limb during locomotion. The relative compliance of the transverse fibers may allow expansion of underlying muscles when they contract.

Chemical composition of human and canine fascia lata.

The fascial system is an integral part of the musculoskeletal system. It is a three-dimensional network of connective tissue spreading ubiquitously throughout the body, surrounding muscles, bones, internal organs, nerves, vessels, and other structures. The basic biophysical properties of the fascial system are determined by its structure and chemical composition. This study aimed to determine the elemental composition of pathologically unchanged fascia lata of the thigh, collected during autopsies on humans and dogs. The wide spectrum of elements analysed included both macro and micro elements. The analyses were conducted using scanning electron microscopy with X-ray microanalysis (SEM-EDS). Concentrations of the following macro and micro elements were determined: C, N, O, Na, Mg, Al, Si, P, S, Cl, K, Ca, Ti, Fe Co, Ni, Cu, and Zn. The obtained results showed significant differences between human and canine fascia lata regarding the content of most of the examined elements (p < 0.05), except for N. These data may in future provide a starting point for the establishment of reference values for the content of various elements in normal fascial tissue and may also serve to verify the usefulness of experimental animal material as a substitute for human tissue.

Regional variability, processing methods, and biophysical properties of human fascia lata extracellular matrix.

This study aims to assess the regional variability, processing methods, mechanical, biochemical, and cellular properties of human fascia lata as a scaffold for soft tissue repair and tissue engineering applications. Ten pairs of fascia lata (donor age 18-55) were used. One fascia patch from each pair was used to assess the geometric and biomechanical variability of fresh fascia. The other from each pair was subjected to 1 of 2 allograft processing methods: antibiotic soak alone or acellularization plus antibiotic soak. Stiffness, modulus, hydroxyproline, chondroitin/dermatan sulfate glycosaminoglycan (CSDS GAG), and DNA content were quantified in fascia from fresh and treated groups. The effect of location was not significant for thickness or stiffness within a 6 x 12 cm2 region of the iliotibial tract of fresh human fascia lata. Processing did not significantly change the stiffness, modulus, or CSDS GAG content of fascia ECM. However, hydroxyproline (collagen) content is significantly reduced in acellularized fascia, probably reflecting a removal of soluble collagen during the treatment (p < 0.02). Processing reduced the DNA content of fresh fascia approximately 10-fold (p < 0.001). The mechanical, chemical and ultrastructural similarities between fascia lata and tendon may make fresh or processed fascia an attractive ECM scaffold for soft tissue, particularly tendon, repair.

Intact genetic material is present in commercially processed cadaver allografts used for pubovaginal slings.

PURPOSE: We determined the presence, quantitated the concentration and assessed the length of DNA present in 4 commercially available human cadaver allografts. MATERIALS AND METHODS: We evaluated 10 tissue samples from each of 4 commercial sources of human allograft (Mentor Corp., Santa Barbara, California; Musculoskeletal Transplant Foundation, Edison, New Jersey; Regeneration Technologies, Inc., Alachua, Florida; and Life Cell Corp., Woodlands, Texas) for intact DNA segments. All allograft samples underwent a standard extraction technique (proteinase K/sodium dodecyl sulfate/phenol) to isolate genetic material. Spectrophotometry evaluation was done to quantify DNA concentrations. Polymerase chain reaction was performed to amplify the retrieved DNA material. Agarose gel electrophoresis was performed to determine the size of DNA fragments. RESULTS: Of the 49 samples tested from all 4 commercial sources of human allograft fascia 39 (97.5%) contained DNA of 400 to 2,000 bp segments. A 400 bp DNA segment was present in 9 Mentor, 10 Musculoskeletal Transplant Foundation, 10 Regeneration Technologies and 10 Life Cell samples. A 700 bp DNA segment was present in 10 Musculoskeletal Transplant Foundation, 10 Regeneration Technologies and 10 Life Cell allografts. A 2,000 bp DNA segment was present in 10 Life Cell tissues. CONCLUSIONS: Intact genetic material was present in all 4 commercially processed human allografts. Tissue processing did not completely eliminate intact DNA segments. The size of the intact DNA and the concentration of DNA varied widely based on tissue processing methods.

Physical and chemical processing for a human dura mater substitute.

OBJECT: Allogenic human fascia lata used in neurosurgery, as dura mater substitute, can be associated with a risk of viral and bacterial transmission. Chemical and physical procedures, developed to inactivate virus and bacteria, have been applied to fascia lata. The aim of this study consists in the evaluation of the biological properties of this treated graft. METHODS: Grafts were treated with solvent detergents, freeze-dried for conservation and gamma irradiated (25,000 Gy) for sterilization. The indirect toxicity evaluation was performed by extraction method, according to the International Standard Organization (ISO). First, the cytotoxic effect of each extracts incubated in the presence of human fibroblasts (WI38) was quantitatively assessed by measuring the cell growth, the viability (succinate dehydrogenase activity, MTT), the membrane integrity (uptake of the neutral red by viable cells, NR) as well as the release of lactate dehydrogenase in the culture medium. Second, confocal laser scanning microscopy (CLSM) was used to assess the direct contact between human primary fibroblasts and graft. CLSM was performed at days 3 and 7 after cells loading. RESULTS: No acute cytotoxicity was observed for chemically processed allografts. Cells loaded on the graft have demonstrated a good growth and spreading. CONCLUSIONS: Human fascia lata secured against conventional and non-conventional agents is a fully biocompatible alternative to the available dural graft materials.

pN collagen type III within tendon grafts used for anterior cruciate ligament reconstruction.

This study measured the amount of immature collagen type III present in tendon rafts obtained from anterior cruciate ligament (ACL) reconstructions. These values were compared with those obtained from control grafts typically used for reconstruction--Achilles, patellar, and fascia lata--and also to the normal ACL. Analyses were performed using a commercially available radioimmunoassay (RIA). The RIA made use of a rabbit polyclonal antibody specific to the amino terminus of procollagen type III. The specificity of the Ab was confirmed by a western blot. Fibril diameter of each of the above samples was measured by transmission electron microscopy (TEM). We thus were able to determine if there was a relationship between pN collagen III content and fibril diameter. The mean amount of pN collagen type III in the normal tendon control group was 0.8 +/- 0.3 ng/microg total protein (range 0.0-2.5 ng/microg). There was significantly greater pN collagen III (16 +/- 3.7 ng/microg total protein) in the grafts containing an average fibril diameter <55 nm than in the normal tendons or ACL (P < 0.05). Grafts with an average fibril diameter >55 nm had similar levels of pN collagen III (1.0 +/- 0.79 ng/microg) as the controls. There was also significantly less pN-collagen III within the functional grafts (5.3 +/- 1.9 ng/microg) as compared to failed grafts, (21.6 +/- 5.1 ng/microg, P < 0.05). These results indicate that incomplete processing of procollagen III may be responsible for some of the ultrastructural alterations seen in tendon grafts. Since ultrastructural organization is believed to influence mechanical properties of these tissues. pN collagen III levels may be a possible indicator of ligament or tendon weakness.

Genetic material is present in cadaveric dermis and cadaveric fascia lata.

PURPOSE: We determined whether genetic material is present in the commercially processed cadaveric allografts used in sling surgery. MATERIALS AND METHODS: We evaluated 16 samples from 2 commercial sources of human allograft, including 8 each of freeze-dried gamma irradiated cadaveric fascia lata and acellular cadaveric dermis. Fresh human rectus fascia and sterile saline served as positive and negative controls, respectively. All samples underwent a standard proteinase K/sodium dodecyl sulfate/phenol extraction technique to isolate DNA. Polymerase chain reaction was done to amplify the retrieved DNA material, spectrophotometry to quantify DNA concentration and agarose gel electrophoresis to determine the size of DNA fragments. RESULTS: Of the 16 samples tested from 2 commercial sources of human allograft fascia 14 (87.5%) contained DNA. Mean DNA concentration plus or minus standard error was 258.3 +/- 80.1 and 272.8 +/- 168.8 microg./gm. tissue for cadaveric fascia lata and cadaveric dermis, respectively. Polymerase chain reaction amplified DNA segments of 2,000 bp from 1 of each of the 8 samples of cadaveric fascia lata and cadaveric dermis. CONCLUSIONS: Freeze-dried gamma irradiated cadaveric fascia lata and acellular cadaveric dermis contained intact DNA.

Dermatan sulfates of normal and scarred fascia.

We evaluated the composition of dermatan sulfates (DS) derived from 23 samples of normal and 23 samples of scarred fascia lata. We analyzed the molecular weight of intact DS chains and the length of chain regions comprising: (1) clusters of L-iduronate-containing disaccharides ("iduronic sections"); (2) clusters of D-glucuronate-containing disaccharides ("glucuronic sections"); and (3) copolymeric sections with both types of disaccharides. A portion of scarred fascia DS chains demonstrated higher molecular weight compared with those from normal tissue. Most disaccharides of DS chains derived from both fascia types form copolymeric segments - heterogeneous in size - with alternatively distributed single disaccharides with glucuronic residues and mainly single ones with iduronate. Only a small number of disaccharides form "glucuronic sections" of heterogeneous size or short "iduronic sections". However, the scarred fascia DS chains demonstrate an increased content of shorter "glucuronic sections" and shorter, often oversulfated, copolymeric segments. It seems that in normal fascia, the DS chain type with a single, long copolymeric region and a single, shorter "glucuronic section" is predominant, while in scarred tissue an increase in multidomain DS chain content may occur.