Collagen Preservation Research Articles

Photoprotective Effects of Ice Plant (Mesembryanthemum crystallinum) Callus Extract on Gene Expression of Human Dermal Fibroblast Against UV Exposure

UV exposure is the principal cause of extrinsic photoaging. Antioxidant-related genes (SOD2 and CAT) and collagen-related genes (COL1A1 and TIMP1) were selected for analysis of the mRNA expression in human dermal fibroblasts (CCD-966SK) using qPCR. In this study, UVA-exposed (15 J/cm2 cells showed decrease in SOD2 , CAT (p < 0 001) and COL1A1 (p < 0 05) gene expression, indicating the decline of antioxidant ability and collagen formation. However, treatment with ice plant callus extract (2 mg/mL, 24 h) before UVA exposure significantly up-regulated SOD2 , CAT , COL1A1 and TIMP1 genes (p < 0 01) by 9.5, 2.7, 1.7 and 3.8 times, respectively, compared with those of the control group, and by 10.2, 4.3 (p < 0 001), 2.1 and 3.8 times, respectively, compared with those of the UVA (only) group. These results demonstrated that ice plant extracts affect both antioxidant- and collagen-related gene expressions, and show positive effects on endogenous antioxidant activity and skin collagen preservation.

Three-Dimensional Culture of Mouse Spermatogonial Stem Cells Using A Decellularised Testicular Scaffold.

Objective Applications of biological scaffolds for regenerative medicine are increasing. Such scaffolds improve cell attachment, migration, proliferation and differentiation. In the current study decellularised mouse whole testis was used as a natural 3 dimensional (3D) scaffold for culturing spermatogonial stem cells. Materials and Methods In this experimental study, adult mouse whole testes were decellularised using sodium dodecyl sulfate (SDS) and Triton X-100. The efficiency of decellularisation was determined by histology and DNA quantification. Masson’s trichrome staining, alcian blue staining, and immunohistochemistry (IHC) were done for validation of extracellular matrix (ECM) proteins. These scaffolds were recellularised through injection of mouse spermatogonial stem cells in to rete testis. Then, they were cultured for eight weeks. Recellularised scaffolds were assessed by histology, real-time polymerase chain reaction (PCR) and IHC. Results Haematoxylin-eosin (H&E) staining showed that the cells were successfully removed by SDS and Triton X-100. DNA content analysis indicated that 98% of the DNA was removed from the testis. This confirmed that our decellularisation protocol was efficient. Masson’s trichrome and alcian blue staining respectively showed that glycosaminoglycans (GAGs) and collagen are preserved in the scaffolds. IHC analysis confirmed the preservation of fibronectin, collagen IV, and laminin. MTT assay indicated that the scaffolds were cell-compatible. Histological evaluation of recellularised scaffolds showed that injected cells were settled on the basement membrane of the seminiferous tubule. Analyses of gene expression using real-time PCR indicated that expression of the Plzf gene was unchanged over the time while expression of Sycp3 gene was increased significantly (P=0.003) after eight weeks in culture, suggesting that the spermatogonial stem cells started meiosis. IHC confirmed that PLZF-positive cells (spermatogonial stem cells) and SYCP3-positive cells (spermatocytes) were present in seminiferous tubules. ConclusionSpermatogonial stem cells could proliferate and differentiated in to spermatocytes after being injected in the decellularised testicular scaffolds.

Optimizing the decellularization process of human maxillofacial muscles for facial reconstruction using a detergent-only approach.

Trauma, congenital diseases, and cancer resection cause muscle deformities of the human facial muscle. Muscle defects are either treated with local or distal flaps if direct closure is not possible. However, such surgical interventions are limited by donor morbidity and limited tissue availability. Decellularized scaffolds provide alternative strategies for replacing and restoring missing facial muscle by creating scaffolds that mimic the native tissue. This study aimed to develop a protocol to decellularize human zygomaticus major muscle (ZMM) and masseter muscle (MM). Three protocols were assessed including a detergent-only treatment (DOT), detergent-enzymatic treatment (DET) protocol, and a third nondetergent nonenzymatic treatment protocol. Scaffolds were then characterized via histological, immunofluorescent, and quantitative techniques to assess which protocol provided optimal decellularization and maintenance of the extracellular matrix (ECM). The results demonstrated three cycles of DOT protocol consisting of 2% sodium dodecyl sulfate for 4 hr was optimal for decellularization for both ZMM and MM. After three cycles, DNA content was significantly reduced compared with native ZMM and MM (p < .05) with preservation of collagen and glycosaminoglycan content and ECM on histological analysis. DET and nondetergent nonenzymatic treatment protocols were unsuccessful in decellularizing the ZMM and MM with residual DNA content after four cycles and caused ECM disruption on histological analysis. All protocols did not impair the mechanical properties and supported human fibroblast growth. In conclusion, the DOT protocol is effective in producing human decellularized muscle scaffolds that maintain the ECM. Further investigation of detergent only decellurization techniques should be explored as a first step to create effective scaffolds for muscle tissue engineering.

Fluorescence screening of collagen preservation in tooth dentine

Isotope analysis of collagen from high-resolution sequential samples of dentine has become a popular tool to provide diachronic insights into individual life histories including childhood diet, stress episodes, and mobility during the tooth formation period. New microsampling approaches improve temporal resolution enabling tracking of diet shifts within relatively short time-spans. These methods, however, necessarily deliver small samples and depend upon good collagen preservation at similar high-resolution scales. Yet present methods indicate state of collagen preservation only after sampling and isotope analysis. Here we present a method for prior determination of collagen preservation, based on differential autofluorescence imaging of tooth longitudinal thin sections to map regions of intact and degraded collagen in the corresponding sampling areas. We find that even in dentine areas that were apparently intact based on histology, decay was detected in fluorescence images and that these could be correlated to lower collagen amounts and higher C/N ratios. Areas identified as ‘severely decayed’ dissolved after demineralization. The presented dual-colour autofluorescence imaging approach allows determination of areas of degraded collagen at high resolution. This can be used to detect diagenetic changes in collagen ahead of sampling and facilitates selection of well-preserved samples.

Bone diagenesis in a Mycenaean secondary burial (Kastrouli, Greece)

This paper presents the characteristics of bone diagenesis in a secondary commingled Mycenaean burial in Kastrouli (Phocis, Greece) through the histological (light microscopy), physical (FTIR-ATR), and biochemical (collagen) analysis of seventeen human (including two petrous bones) and seven animal bones. Post-mortem modifications in bone microstructure, bioapatite, and collagen were characteristic of burial environments with seasonal groundwater and temperature fluctuations. The two human petrous bones displayed a lack of microscopic focal destruction (MFD) sites and a generally good histological preservation, but although a small sample size, did not show any better bioapatite and collagen preservation compared with human femora. Intra-site variations were defined by three main diagenetic patterns that display differences in histological modifications, crystallinity changes, and collagen degradation. These different patterns were either related to different microenvironment conditions and/or influenced by possible differences in the early taphonomic histories experienced by bones prior to secondary deposition. Further, this study highlights the importance of infrared splitting factor (IRSF), carbonate/phosphate ratio (C/P), and general histological index (GHI) for the qualitative assessment of archaeological bone, and the potential use of amide/phosphate ratio (Am/P) as a collagen predictor.

Bone diagenesis in a Mycenaean secondary burial (Kastrouli, Greece)

This paper presents the characteristics of bone diagenesis in a secondary commingled Mycenaean burial in Kastrouli (Phocis, Greece) through the histological (light microscopy), physical (FTIR-ATR), and biochemical (collagen) analysis of seventeen human (including two petrous bones) and seven animal bones. Post-mortem modifications in bone microstructure, bioapatite, and collagen were characteristic of burial environments with seasonal groundwater and temperature fluctuations. The two human petrous bones displayed a lack of microscopic focal destruction (MFD) sites and a generally good histological preservation, but although a small sample size, did not show any better bioapatite and collagen preservation compared with human femora. Intra-site variations were defined by three main diagenetic patterns that display differences in histological modifications, crystallinity changes, and collagen degradation. These different patterns were either related to different microenvironment conditions and/or influenced by possible differences in the early taphonomic histories experienced by bones prior to secondary deposition. Further, this study highlights the importance of infrared splitting factor (IRSF), carbonate/phosphate ratio (C/P), and general histological index (GHI) for the qualitative assessment of archaeological bone, and the potential use of amide/phosphate ratio (Am/P) as a collagen predictor.

Extracellular matrix electrospun membranes for mimicking natural renal filtration barriers

Kidney diseases are recognized as a major health problem, which affect 10% of the population. Because currently available therapies have many limitations, some tissue engineering strategies have been emerging as promising approaches in this field.In this work, porcine kidneys were decellularized to obtain decellularized kidney extracellular matrix (dKECM).11dKECM (decellularized kidney extracellular matrix) Our results demonstrate a successful protocol of decellularization characterized by the removal of nucleic acid material and preservation of collagen and glycosaminoglycans. Blends of polycaprolactone (PCL)22PCL (polycaprolactone) and dKECM were prepared by electrospinning and characterized. The biological performance of the membranes was tested with a human kidney cell line (HK-2) for 7 days. It was observed that cellular metabolic activity, proliferation and protein content increased with an increase in dKECM concentrations (30, 50 and 70%). Additionally, the expression of zona occludens-1 was revealed on dKECM-containing membranes but not on pure PCL membranes.To the best of our knowledge this is the first time that natural extracellular matrix is used to mimic the kidney basement membrane as an in vitro model. This could be a valuable tool for regenerative nephrology and may have an impact on the development of kidney advanced therapies in the future.

Portable FTIR for on-site screening of archaeological bone intended for ZooMS collagen fingerprint analysis

Faunal remains play an important role in helping reconstruct Palaeolithic hunter-gatherer subsistence and mobility strategies. However, differential bone preservation is an issue in southern European prehistoric sites, which often makes morphological identification impossible. Zooarchaeology by Mass Spectrometry (ZooMS) is a new, low-cost method that can improve NISP statistical significance in a replicable way by using diagnostic peptides of the dominant collagen protein as a fingerprint of animal (including hominin) species. It is also a powerful tool to assess collagen preservation for radiocarbon dating. This paper presents the proof of concept of a method for evaluating collagen preservation in a quick and minimally destructive way in the field prior to ZooMS analysis by using a portable Fourier Transform Infrared Spectrometer (FTIR) equipped with an attenuated total reflectance accessory (ATR). The method was tested on faunal assemblages from two north-western Italian sites: Riparo Bombrini and Arma Veirana. Both are important sites for understanding the Middle and Upper Palaeolithic of Liguria but are located in two distinct environmental contexts (coast vs. mountainous hinterland) that impacted collagen preservation. The relative abundance of collagen in powdered bone samples was evaluated by calculating the Amide I to phosphate ratio (CO/P) from spectra collected with two portable and three laboratory-based FTIR instruments. The bones were then analysed by ZooMS and the results were compared to evaluate the effectiveness of using FTIR as a screening technique. Results indicate that FTIR instruments are excellent tools to predict collagen preservation in fragmented archaeological bones, but a screening method should be first calibrated on the analysed faunal assemblage by conducting a test of the CO/P screening procedure with various measurement systems on a single FTIR instrument.

Advances in bone preservation: Identifying possible collagen preservation using sulfur speciation mapping

The complexity and heterogeneity of bone chemistry makes it difficult to discern information on physiological and taphonomic processes stored within the bone matrix. Analysis of archaeological and palaeontological bone becomes more difficult because in many cases the most pivotal specimens are too scientifically valuable for destructive analysis. This problem is further escalated by the fact that the heterogeneity of the bone may cause small “pockets” of preservation that can be missed during sampling. Therefore, a non-destructive technique that can spatially resolve such heterogeneity within the bone is needed. Here we use microfocus, non-destructive synchrotron-based X-Ray Fluorescence (XRF) imaging and X-ray Absorption Spectroscopy (XAS) to map the organic constituents within extant and fossil bovid bones. XAS analysis of sulfur allowed organic sulfur (within collagen as methionine) to be distinguished from inorganic sulfate (within bone apatite). Mapping and quantification of organic sulfur within the samples were made by setting the beam to the methionine resonance, allowing for the detection, distribution and quantification of collagen present by using organic sulfur as an internal marker. Results show organic sulfur to be distributed in small “pockets” throughout the bone matrix in both extant and fossil specimens. Significant loss of organic sulfur was seen in specimens between 100 ka and 650 ka with little organic sulfur preservation persisting after this date. Comparison of residual organic sulfur concentrations as a function of sample age revealed a second order rate law for organic sulfur oxidation (k ≈ 1 × 10−5 y−1) within bone. These results show that non-destructive, synchrotron-based XRF mapping of organic sulfur is a useful tool for not only calculating rates of collagen degradation through time, but also identifying areas of potential collagen preservation for other paleobiological applications such as proteomics and stable isotope analyses.

Intratumoral collagen index predicts mortality and survival in canine cutaneous mast cell tumours.

Mast cell tumours (MCTs) constitute almost 25% of cutaneous neoplasms in dogs. Their biological behaviour is predicted using histopathological grading which is based on several subjective criteria that are vulnerable to intra- and interobserver variability. To improve the prediction of the biological behaviour, several complementary markers have been studied. The integrity of the extracellular matrix (ECM) may play a protective role against tumoral progression, and favour cellular proliferation, angiogenesis, invasion and metastases when altered. To evaluate the quantification of collagen and elastic fibres as prognostic markers for MCTs. Thirty-eight random cases of canine cutaneous MCT surgically treated with wide margins were included. Intratumoral collagen and elastic fibres were identified and quantified on histological sections stained with Masson's trichrome, Picrosirius red and Verhoeff; the results were compared with histopathological grades, mortality due to the disease and postsurgical survival. Morphometric analysis revealed a significant relationship between histopathological grade and intratumoral collagen index (CoI). In addition, the CoI was considered an independent indicator for mortality and postsurgical survival. These results support the importance of the CoI in the grading and prognosis of MCTs, suggesting that preservation and/or synthesis of collagen have the potential to become targets for MCT therapeutics.

The earliest burial from the Caribbean coast of Nicaragua: the Angi shell-matrix site

Abstract

Exploring the potential of human bone and teeth collagen from Prehistoric Cyprus for isotopic analysis

This pilot study attempts to document the potential of Prehistoric human bone and teeth collagen from Cyprus (9th-2nd mill. BC), for isotopic analysis and palaeodietary reconstruction. We sampled archaeological human skeletons and some faunal remains coming from six sites located in different locations, with different burial modes. The analysis of carbon and nitrogen elemental compositions and stable isotope ratios (δ13C, δ15N), indicate an extremely poor preservation of collagen, probably in relationship with burying conditions. Although very few individuals were successfully analysed, stable isotope data from this study allow a discussion on different protein food resources intake by humans in comparison with some other published data in the Near East (Greece, Cyprus, Turkey) from the Neolithic to the Bronze Age. These diachronic data provide documentations for future studies, including palaeodietary and environmental field research.

Use of micro-computed tomography imaging and porosity measurements as indicators of collagen preservation in archaeological bone

Collagen isolated from archaeological bone is a common material for radiocarbon dating, stable isotope analysis, and zooarchaeology by mass spectrometry (ZooMS). However, not all bones contain extant collagen, leading to unnecessary destruction of unproductive bones and wasted laboratory time and resources. An aim of this research is to study bone diagenesis, particularly collagen destruction, in an effort to develop a minimally destructive method for identifying bones with high collagen content. In a multi-method study of variably preserved bones from Etton, Cambridgeshire, UK, we examined material properties of Neolithic cattle and sheep bones including porosity, surface area, and elemental composition. Micro-computed tomography (microCT) is an imaging technique that furnishes three-dimensional images of mineralized materials such as bone. Cortical bone porosity, the percentage of total bone volume consisting of empty space as calculated using microCT, can act as a proxy for bone collagen preservation. In general, bones with high cortical porosity are unlikely to contain sufficient collagen for further analysis. Bones with apparently low cortical porosity have a more varied range of collagen preservation. Bone samples with low porosity and no extant collagen often contain micropores with a diameter of 10 nm or less that cannot be seen in microCT images but are apparent in pore size distributions measured by mercury porosimetry, and indicated by high surface areas measured by nitrogen adsorption. Furthermore, a re-evaluation of light-induced breakdown spectroscopy data from this same assemblage confirms that ratios of calcium to fluorine may likewise indicate the state of diagenesis.

BMP-2 and type I collagen preservation in human deciduous teeth after demineralization.

Great interest has recently been focused on tooth and tooth derivatives as suitable substrates for the treatment of alveolar bone defects. Here, we propose the use of demineralized baby teeth (BT) as potential grafting materials for bone augmentation procedures. Particles of human BT (Ø < 1 mm) were demineralized by means of a chemical/thermal treatment. Demineralized BT particles were thoroughly characterized by scanning electron microscopy/energy dispersive X-ray analyses to evaluate the effects of the demineralization on BT topography and mineral phase composition, and by enzyme-linked immunosorbent assays (ELISA) to quantify collagen and bone morphogenetic protein-2 (BMP-2) protein contents. The response of SAOS-2 cells to exogenous BMP-2 stimulation was evaluated to identify the minimum BMP-2 concentration able to induce osteodifferentiation in vitro (alkaline phosphatase (ALP) activity). The demineralization treatment led to a dramatic decrease in relative Ca and P content (%) of ≈75% with respect to the native BT particles, while preserving native protein conformation and activity. Interestingly, the demineralization process led to a rise in the bioavailability of BMP-2 in BT particles, as compared to the untreated counterparts. The BMP-2 content found in demineralized BT was also proved to be very effective in enhancing ALP activity, thus in the osteodifferentiation of SAOS-2 cells in vitro, as confirmed by cell experiments performed upon exogenously added BMP-2. In this study we demonstrate that the BMP-2 content found in demineralized BT is very effective in inducing cell osteodifferentiation, and strengthens the idea that BTs are very attractive bioactive materials for bone-grafting procedures.

Impact of different intratracheal flows during lung decellularization on extracellular matrix composition and mechanics.

To evaluate different intratracheal flow rates on extracellular matrix content and lung mechanics in an established lung decellularization protocol. Healthy mice were used: 15 for decellularization and five to serve as controls. Fluids were instilled at 5, 10and 20ml/min flow rates through tracheal cannula and right ventricular cavity (0.5ml/min) in all groups. The 20ml/min rate better preserved collagen content in decellularized lungs. Elastic fiber content decreased at 5 and 10ml/min, but not at 20ml/min, compared with controls. Chondroitin, heparan and dermatan content was reduced after decellularization. An intratracheal flow rate of 20ml/min was associated with lower resistance and greater preservation of collagen to that observed in ex vivo control lungs.

1163 Decorin is associated with preservation of dermal collagen in UVB exposed mice

1163 Decorin is associated with preservation of dermal collagen in UVB exposed mice

Nitrogen content variation in archaeological bone and its implications for stable isotope analysis and radiocarbon dating

The collagen component of ancient bones is routinely isolated for radiocarbon dating and stable isotope studies. However, it is impossible to tell the state of collagen preservation from visual inspection of bones. At the Oxford Radiocarbon Accelerator Unit (ORAU), the percent nitrogen by weight (%N) of a ∼5 mg sample of bone powder is measured on a mass spectrometer and used as a proxy for protein content. A previous study showed that samples with %N > 0.76 are considered likely to produce sufficient collagen for radiocarbon dating (Brock et al., 2010b). However, the extent of variation between bone %N and collagen yield is unclear, as is the intra-bone variation in %N. Here, we report a series of tests performed on Palaeolithic bones known to have variable collagen preservation. This new study shows significant variation in %N within the same bone and that there is sometimes a lack of correlation between %N and collagen yield. These results suggest that for bone samples from difficult environments or from Pleistocene contexts, it may be worth sub-sampling for %N in different locations of the bone (if possible) and then attempting to extract collagen from marginally preserved bones (%N around 0.2–0.7%), as they may still yield sufficient collagen for isotope and dating studies.

Ageing and preservation of HgS-enriched ancient human remains deposited in confinement

Mankind has been exposed to mercury (Hg) for thousands of years, but little information on the role of Hg on the preservation of ancient human remains has been addressed. In particular, the Maya people were expose to cinnabar (HgS), a naturally occurring mineral. Because in the Maya culture the red colour was symbolic of blood and blood sacrifice and associated to death and rebirth, For these reason, they often decorated burial chambers and covered sarcophagi, and corpses of important members of their society with HgS. However, scarce information has become available on the effects of HgS on the preservation of Maya human remains. This paper reports on the preservation of HgS-human remains, kept in sarcophagi away from sunlight and environmental humidity. Two HgS-enriched human remains collected from the Temples of the Scriptures (TS) and Temple XIII (TXIII) in the archaeological site of Palenque, Chiapas, Mexico were studied by radiocarbon dating, high resolution scanning and transmission electron microscopy, and energy dispersive X-ray spectroscopy. Conventional radiocarbon ages for S-TS and S-TXIII were 1360 ± 30 CE and 3100 ± 30 BCE, respectively, evidencing difference in age between the archaeological remains proximal to 1740 yrs. The more ancient specimen, S-TXIII, was linked to the excellent preservation of collagen preservation of collagen fibrils. The more recent remains (S-TS) displayed more abundant and richly diverse biological components, including highly porous tubules with irregular low and high accumulation of Hg in the walls, and ovoid protuberances. Micrographs showed excellently preserved biotubules denoting calcite permineralization, however, the presence of Hg provoked mineral dissolution explained in terms of biological activity. Mercury was found abundantly in ancient bone, which was explained because the dissolution of HgS after interacting with bioorganic material, and the formation of Hg-organic complexes. S-TS showed trace amounts of Hg and S, which were attributed to metabolic activity of microorganisms causing Hg volatilization and S decomposition. Furthermore, although microelemental analysis for both specimens showed the association of Hg with S, and HgS to be the source of these elements, the distribution of Hg clearly showed that the two royal characters held different lifestyle. On the one hand, Hg distribution in bone for the specimens showed that S-TXIII contained Hg in the regions covered by HgS; on the other hand, S-TS contained Hg throughout the bone structure. These results were interpreted to mean that both specimens S-TXII were covered by S-TS after dead and in addition, S-TS belonged in life to a character who was also exposed to Hg via inhalation or consumption of contaminated food, or liquids.

Study of tensiometric properties, microbiological and collagen content in nile tilapia skin submitted to different sterilization methods.

Tissue bioengineering development is a global concern and different materials are studied and created to be safe, effective and with low cost. Nile Tilapia skin had shown its biological potential as covers for the burn wound. This study evaluates the tilapia skin histological, collagen properties and tensiometric resistance, after treatment by different sterilization methods. Tilapia skin samples were submitted to two sterilization processes: (1) chemical, which consisted in two 2% chlorhexidin baths, followed by sequential baths in increasing glycerol concentrations; and (2) radiation, when glycerolized skin samples were submitted to gamma radiation at 25, 30 and 50kGy. Microscopic analyzes were performed through Haematoxylin-eosin and Picrosirius Red under polarized light. For tensiometric analysis, traction tests were performed. Glycerol treated skin presented a discrete collagen fibers disorganization within the deep dermis, while irradiated skin did not show any additional change. Throughout the steps of chemical sterilization, there was a higher proportion of collagen with red/yellow birefringence (type I) in the skin samples up to the first bath in chlorhexidin, when compared to samples after the first two glycerol baths (P<0.005). However, there was no difference in relation to total collagen between groups. In irradiated skin, there was a larger total collagen preservation when using until 30kGy (P<0.005). Tensiometric evaluation did not show significant differences in relation to maximum load in the groups studied. We concluded that chemical and radiation (25 and 30kGy) are efficient methods to sterilize Nile Tilapia skin without altering its microscopic or tensiometric characteristics.

Second-harmonic generation imaging of collagen in ancient bone

Second-harmonic generation imaging (SHG) captures triple helical collagen molecules near tissue surfaces. Biomedical research routinely utilizes various imaging software packages to quantify SHG signals for collagen content and distribution estimates in modern tissue samples including bone. For the first time using SHG, samples of modern, medieval, and ice age bones were imaged to test the applicability of SHG to ancient bone from a variety of ages, settings, and taxa. Four independent techniques including Raman spectroscopy, FTIR spectroscopy, radiocarbon dating protocols, and mass spectrometry-based protein sequencing, confirm the presence of protein, consistent with the hypothesis that SHG imaging detects ancient bone collagen. These results suggest that future studies have the potential to use SHG imaging to provide new insights into the composition of ancient bone, to characterize ancient bone disorders, to investigate collagen preservation within and between various taxa, and to monitor collagen decay regimes in different depositional environments.