Enhanced osteogenicity of the demineralized bone-dermal matrix composite by the optimal partial demineralization for sustained release of bioactive molecules.

Allogenic demineralized bone matrix (DBM), processed to expose bioactive proteins imbedded by calcium salts, is widely used for bone repair and regeneration as an alternative to the autologous bone graft. However, demineralized bone matrices from tissue banks vary significantly in residual calcium content and osteogenicity for clinical bone regeneration. The present study produced DBM with various residual calcium contents by partial demineralization using ethylenediaminetetraacetic acid disodium (EDTA) and hydrochloric acid. Compositional analysis reveals that, as the percent weight loss of bone materials increases from 0% to 74.9% during demineralization, the residual calcium content of DBM decreases from 24.8% to 0.2% and collagen content increases from 29.7% to 92.6%. Calorimetrical analysis and Fourier transform infrared (FTIR) analysis demonstrated that demineralization to the residual calcium content of <4% enables the complete exposure and/or release of bone collagen fibers and other bioactive molecules. In order to evaluate the relationship between the extent of demineralization and the osteogenicity of DBM, DBM particles were fabricated with the aid of acellular dermal matrix (ADM) microfibers to form flexible foam-like DBM/ADM composites. Proteomic analysis identified various type collagens and bone formation-related bioactive molecules in both ADM and DBM. Using the rat bilateral Φ = 5 mm calvarium defect repair model, the study had shown that the DBM/ADM composite with ~20% DBM residual calcium (e.g., ~40% calcium being removed) maximized the osteogenicity for bone defect repair after 4 and 8 weeks. DBM with ~40% calcium removal had the maximal osteogenicity presumably through the sustained release of bioactive molecules during the process of bone regeneration.

Auxiliary Diagnosis of Papillary Thyroid Carcinoma Based on Spectral Phenotype.

Thyroid cancer, a common endocrine malignancy, is one of the leading death causes among endocrine tumors. The diagnosis of pathological section analysis suffers from diagnostic delay and cumbersome operating procedures. Therefore, we intend to construct the models based on spectral data that can be potentially used for rapid intraoperative papillary thyroid carcinoma (PTC) diagnosis and characterize PTC characteristics. To alleviate any concerns pathologists may have about using the model, we conducted an analysis of the used bands that can be interpreted pathologically. A spectra acquisition system was first built to acquire spectra of pathological section images from 91 patients. The obtained spectral dataset contains 217 spectra of normal thyroid tissue and 217 spectra of PTC tissue. Clinical data of the corresponding patients were collected for subsequent model interpretability analysis. The experiment has been approved by the Ethics Review Committee of the Wuhu Hospital of East China Normal University. The spectral preprocessing method was used to process the spectra, and the preprocessed signal respectively optimized by the first and secondary informative wavelengths selection was used to develop the PTC detection models. The PTC detection model using mean centering (MC) and multiple scattering correction (MSC) has optimal performance, and the reasons for the good performance were analyzed in combination with the spectral acquisition process and composition of the test slide. For model interpretable analysis, the near-ultraviolet band selected for modeling corresponds to the location of amino acid absorption peak, and this is consistent with the clinical phenomenon of significantly lower amino acid concentrations in PTC patients. Moreover, the absorption peak of hemoglobin selected for modeling is consistent with the low hemoglobin index in PTC patients. In addition, the correlation analysis was performed between the selected wavelengths and the clinical data, and the results show: the reflection intensity of selected wavelengths in normal cells has a moderate correlation with cell arrangement structure, nucleus size and free thyroxine (FT4), and has a strong correlation with triiodothyronine (T3); the reflection intensity of selected bands in PTC cells has a moderate correlation with free triiodothyronine (FT3).

Integrated transcriptomics and proteomics assay identifies the role of FCGR1A in maintaining sperm fertilization capacity during semen cryopreservation in sheep.

Semen cryopreservation is a promising technology employed in preserving high-quality varieties in animal husbandry and is also widely applied in the human sperm bank. However, the compromised qualities, such as decreased sperm motility, damaged membrane structure, and reduced fertilization competency, have significantly hampered the efficient application of this technique. Therefore, it is imperative to depict various molecular changes found in cryopreserved sperm and identify the regulatory network in response to the cryopreservation stress. In this study, semen was collected from three Chinese Merino rams and divided into untreated (fresh semen, FS) and programmed freezing (programmed freezing semen, PS) groups. After measuring different quality parameters, the ultra-low RNA-seq and tandem mass tag-based (TMT) proteome were conducted in both the groups. The results indicated that the motility (82.63% ± 3.55% vs. 34.10% ± 2.90%, p < 0.05) and viability (89.46% ± 2.53% vs. 44.78% ± 2.29%, p < 0.05) of the sperm in the FS group were significantly higher compared to those in the PS group. In addition, 45 upregulated and 291 downregulated genes, as well as 30 upregulated and 48 downregulated proteins, were found in transcriptomics and proteomics data separately. Moreover, three integrated methods, namely, functional annotation and enrichment analysis, Pearson's correlation analysis, and two-way orthogonal partial least squares (O2PLS) analysis, were used for further analysis. The results suggested that various differentially expressed genes and proteins (DEGs and DEPs) were mainly enriched in leishmaniasis and hematopoietic cell lineage, and Fc gamma receptor Ia (FCGR1A) was significantly downregulated in cryopreserved sperm both at mRNA and protein levels in comparison with the fresh counterpart. In addition, top five genes (FCGR1A, HCK, SLX4, ITGA3, and BET1) and 22 proteins could form a distinct network in which genes and proteins were significantly correlated (p < 0.05). Interestingly, FCGR1A also appeared in the top 25 correlation list based on O2PLS analysis. Hence, FCGR1A was selected as the most potential differentially expressed candidate for screening by the three integrated multi-omics analysis methods. In addition, Pearson's correlation analysis indicated that the expression level of FCGR1A was positively correlated with sperm motility and viability. A subsequent experiment was conducted to identify the biological role of FCGR1A in sperm function. The results showed that both the sperm viability (fresh group: 87.65% ± 4.17% vs. 75.8% ± 1.15%, cryopreserved group: 48.15% ± 0.63% vs. 42.45% ± 2.61%, p < 0.05) and motility (fresh group: 83.27% ± 4.15% vs. 70.41% ± 1.07%, cryopreserved group: 45.31% ± 3.28% vs. 35.13% ± 2.82%, p < 0.05) were significantly reduced in fresh and frozen sperm when FCGR1A was blocked. Moreover, the cleavage rate of embryos fertilized by FCGR1A-blocked sperm was noted to be significantly lower in both fresh (95.28% ± 1.16% vs. 90.44% ± 1.56%, p < 0.05) and frozen groups (89.8% ± 1.50% vs. 82.53% ± 1.53%, p < 0.05). In conclusion, our results revealed that the downregulated membrane protein FCGR1A can potentially contribute to the reduced sperm fertility competency in the cryopreserved sheep sperm.

Comparative proteomic analysis of regenerative acellular matrices: The effects of tissue source and processing method.

Acellular tissue matrices are used in regenerative medicine from weak tissue re-enforcement to cosmetic augmentation. However, proteomic signatures leading to different clinical outcomes among matrices are not well understood. In an attempt to investigate the effects of tissue source and processing method, we examined by liquid chromatography tandem mass spectrometry (LC-MS/MS) the proteomic profiles of 12 regulatory agency-approved acellular matrices (AlloMax, AlloDerm, CollaMend, Heal-All, JayyaLife, ReGen, Renov, Strattice, SurgiMend, Surgisis, UniTrump and Vidasis). The compositions of acellular matrices varied greatly with the number of identified proteins ranging from 7 to 106. The content of individual proteins was between 0.0001% and 95.8% according to their abundances measured by the M/Z signal intensities. Most acellular matrices still contained numerous cellular proteins. AlloMax, AlloDerm, ReGen, Strattice, SurgiMend and Surgisis retained necessary structural and functional proteins to form the extracellular protein-protein interaction networks for cell adhesion, proliferation and tissue regeneration, whereas CollaMend, Heal-All, JayyaLife, Renov, UniTrump and Vidasis had only retained certain structural collagens. Principal component analysis showed that proteomic variations among acellular matrices were largely attributed to tissue source and processing method. Differences in proteomic profiles among acellular matrices offers insights into molecular interpretation for different clinical outcomes, and can serve as useful references for rational design of regenerative bio-scaffolds.

Complete proteomic profiling of regenerative bio-scaffolds with a two-step trypsinization method.

Regenerative bio-scaffolds, widely used for clinical tissue reconstruction and tissue repairs, are functionally diversified and structurally complex decellularized tissue materials (e.g., extracellular matrix, ECM). ECM is naturally cross-linked and can be further selectively cross-linked upon processing. Identification, quantification and bioinformatics functional comparison of all ECM proteins are challenging for regenerative bio-scaffolds. In this study, we have applied proteomic profiling with a two-step sequential trypsinization method, and identified and quantified 300-400 constituent proteins in three commercially available regenerative bio-scaffolds (BioDesign Surgisis, ReGen tissue matrix, and ThormalGEN mesh). These proteins were classified into four categories and 14 subcategories based on their mainly biological function. The main components of regenerative bio-scaffolds were highly abundant ECM structural proteins, and the minor parts of bio-scaffolds were lowly abundant, less cross-linked, functionally more diversified proteins, especially extracellular fluid proteins that were easily solubilized by trypsin. The comparative analysis has revealed large differences in the number, type, abundance and function of identified proteins, as well as the extent of decellularization and cross-linking among regenerative bio-scaffolds. So, the proteomic profiling with a two-step sequential trypsinization method could not only provide the molecular basis to better understand the degradation process of regenerative bio-scaffolds in vivo and different clinical outcomes among various regenerative bio-scaffolds, facilitate the exploration of the response mechanisms in the host's early clinical stages of ECM-induced tissue regeneration that is still poorly understood, but also can be used for optimization of the decellularization and cross-linking process, product characterization and rational design of new ECM products.

Cryopreservation did not affect sperm DNA methylation levels of genes related to fertilization and embryonic development of cynomolgus macaque (Macaca fascicularis).

DNA methylation alters gene expression in numerous biological processes, including embryonic development. It is little known about the effect of cryopreservation on sperm DNA methylation. The present study has investigated whether cryopreservation causes abnormal DNA methylation in cynomolgus macaque sperm for five critical genes that includes the maternally imprinted gene (SNRPN), genes associated with male infertility (HSPA1L, MTHFR) and genes involved in embryonic development (TET3, LZTR1). Our results showed that sperm motility, the percentage of acrosomal integrity, DNA integrity and mitochondrial membrane potential were decreased after cryopreservation either being frozen with penetrating cryoprotectant, glycerol (Gly) or ethylene glycol (EG), compared to fresh sperm (p = 0.000), but the methylation patterns of the five target genes from cynomolgus macaque sperm samples were not affected after cryopreservation as evaluated by the Bisulfite Sequencing PCR (BSP) method. The data indicates that the current protocol for sperm cryopreservation of cynomolgus macaque is safe in terms of DNA methylation levels in these genes related to critical sperm functions.

Identifying the heat resistant genes by multi-tissue transcriptome sequencing analysis in Turpan Black sheep.

Heat stress not only affects the physical condition but also affects reproductive performance in sheep. A thorough understanding of the molecular and physiological mechanisms underlying heat stress would certainly improve livestock productivity and provide genetic evaluation ways for heat resistant breeds selection. In this study, 85 Turpan Black sheep, a breed exhibited excellent heat resistance from long-term artificial selection, and 85 heat sensitive Kazakh sheep in Turpan basin were tested for physiological and reproductive performance from July to August in summer. The results showed that the estrus rate was significantly higher in Turpan Black sheep (P < 0.05), while the heart rate and respiratory rate of Turpan Black sheep are significantly lower than that of Kazakh sheep (P < 0.05). Furthermore, to clarify genes participated in heat stress response, the pituitary, ovarian and hepatic tissues from three Turpan Black sheep and three Kazakh sheep were subjected to RNA-seq. The results indicated that 32, 49 and 69 genes were up-regulated, and 39, 60 and 145 genes were down-regulated in pituitary, ovarian and hepatic tissues in Turpan Black sheep compared with that of the Kazakh sheep, respectively. KEGG and gene set enrichment analysis showed that the differentially expressed genes were mainly involved in signal transduction pathways. In particular, the differentially expressed genes in hepar were enriched in the energy metabolism pathway, while the differentially expressed genes in ovarian tissue were enriched in the ovarium steroidogenesis pathway. In conclusion, our results implied that the pituitary-ovary axis might include hepar as downstream targeted organism in heat resistant regulation. Under heat stress, the signals released from pituitary would impact steroidogenesis in ovary, and further alter energy metabolism in hepar. As we know, this is the first comparative study to investigate the gene expression in multi-tissue in sheep under heat stress.

Improving Sperm Cryopreservation With Type III Antifreeze Protein: Proteomic Profiling of Cynomolgus Macaque (Macaca fascicularis) Sperm.

Antifreeze protein III (AFP III) is used for the cryopreservation of germ cells in various animal species. However, the exact mechanism of its cryoprotection is largely unknown at the molecular level. In this study, we investigated the motility, acrosomal integrity, and mitochondrial membrane potential (MMP), as well as proteomic change, of cynomolgus macaque sperm after cryopreservation. Sperm motility, acrosomal integrity, and MMP were lower after cryopreservation (p < 0.001), but significant differences in sperm motility and MMP were observed between the AFP-treated sperm sample (Cryo+AFP) and the non-treated sample (Cryo-AFP) (p < 0.01). A total of 141 and 32 differentially expressed proteins were, respectively, identified in cynomolgus macaque sperm cryopreserved without and with 0.1 µg/ml AFP III compared with fresh sperm. These proteins were mainly involved in the mitochondrial production of reactive oxygen species (ROS), glutathione (GSH) synthesis, and cell apoptosis. The addition of AFP III in the sperm freezing medium resulted in significant stabilization of cellular molecular functions and/or biological processes in sperm, as illustrated by the extent of proteomic changes after freezing and thawing. According to the proteomic change of differentially expressed proteins, we hypothesized a novel molecular mechanism for cryoprotection that AFP III may reduce the release of cytochrome c and thereby reduce sperm apoptosis by modulating the production of ROS in mitochondria. The molecular mechanism that AFP III acts with sperm proteins for cellular protection against cryoinjuries needs further study.

Evaluation of a novel tilapia-skin acellular dermis matrix rationally processed for enhanced wound healing.

Acellular Dermal Matrix (ADM) is mainly made with human or porcine skins and has the risk of zoonotic virus transmission. The fish skin-derived ADM could overcome the shortcoming. Fish skin acellular matrix has been used as wound dressing, but there is few systematic studies on tilapia-skin acellular dermal matrix (TS-ADM). In the present study, a novel TS-ADM was made by an alkaline decellularization process and γ-irradiation. The physical properties, biocompatibility, pre-clinical safety and wound healing activity of TS-ADM were systematically evaluated for its value as a functionally bioactive wound dressing. Histopathological analysis (hematoxylin and eosin staining, 4,6-diamidino-2-phenylindole (DAPI) staining) and DNA quantification both proved that the nuclear components of tilapia skin were removed sufficiently in TS-ADM. Compared to the commercial porcine acellular dermal matrix (DC-ADM), TS-ADM has distinctive features in morphology, thermal stability, degradability and water vapor transmission. TS-ADM was more readily degradable than DC-ADM in vitro and in vivo. In both rat and mini-pig skin wound healing experiments, TS-ADM was shown to significantly promote granulation growth, collagen deposition, angiogenesis and re-epithelialization, which may be attributed to the high expression of transforming growth factor-beta 1 (TGF-ß1), alpha-smooth muscle actin (α-SMA) and CD31. Herein, the novel TS-ADM, used as a low-cost bioactive dressing, could form a microenvironment conducive to wound healing.

DSC Analysis of Thermophysical Properties for Biomaterials and Formulations.

The development of freezing and freeze-drying processes for biological samples requires knowledge of the thermophysical properties of the biomaterial and protectant solutions involved. This chapter provides an introduction on the use of differential scanning calorimetry (DSC) to study thermophysical properties of biomaterials in protective solutions. It covers specific methods to study thermal events related to freezing and drying processes including crystallization, eutectic formation, glass transition, devitrification, recrystallization, melting, molecular relaxation, and phase separation.

Evaluation and preservation of vascular architectures in decellularized whole rat kidneys.

Organ transplantation is the gold standard treatment for end-stage organ failure. Due to the severe shortage of transplantable organs, only a tiny fraction of patients may receive timely organ transplantation every year. Decellularization-recellularization technology using allogeneic and xenogeneic organs is currently conceived to be a promising solution to generate functionally transplantable organs in vitro. This approach, however, still faces tremendous technological challenges, one of them being the ability to evaluate and preserve the integrity of vascular architectures upon decellularization and cryostorage of the whole organ matrices so that the off-the-shelf organ grafts are available on demand for clinical applications. In the present study, we report a Micro-CT imaging method for evaluating the integrity of vasculature of the decellularized whole organ scaffolds with/without freezing/thawing. The method uses radiopaque Microfil perfusion and x-ray fluoroscopy to acquire high-resolution angiography of the organ matrix. The whole rat kidney is decellularized using a new multistep perfusion protocol with the combined use of Triton X-100 and DNase. The decellularized kidney matrix is then cryopreserved after the pretreatment with different cryoprotectant solutions. The reconstructed tomographic images from Micro-CT confirm various structural alterations in the vasculature of the whole decellularized kidney matrix with/without frozen storage. The freezing damage to the vascular architectures can be reduced by perfusing cryoprotectant solutions into the whole kidney matrix. Ice-free cryopreservation with the vitrification solution VS83 can successfully preserve the integrity of the whole kidney matrix's vasculature after frozen storage.

Assembly and Function of a Bioengineered Human Liver for Transplantation Generated Solely from Induced Pluripotent Stem Cells.

The availability of an autologous transplantable auxiliary liver would dramatically affect the treatment of liver disease. Assembly and function in vivo of a bioengineered human liver derived from induced pluripotent stem cells (iPSCs) has not been previously described. By improving methods for liver decellularization, recellularization, and differentiation of different liver cellular lineages of human iPSCs in an organ-like environment, we generated functional engineered human mini livers and performed transplantation in a rat model. Whereas previous studies recellularized liver scaffolds largely with rodent hepatocytes, we repopulated not only the parenchyma with human iPSC-hepatocytes but also the vascular system with human iPS-endothelial cells, and the bile duct network with human iPSC-biliary epithelial cells. The regenerated human iPSC-derived mini liver containing multiple cell types was tested in vivo and remained functional for 4 days after auxiliary liver transplantation in immunocompromised, engineered (IL2rg-/-) rats.

Effects of Me2SO and Trehalose on the Cell Viability, Proliferation, and Bcl-2 Family Gene (BCL-2, BAX, and BAD) Expression in Cryopreserved Human Breast Cancer Cells.

Long-term cryopreservation of the viability and metabolic state of cells in cancer cell/tissue specimens has significant implications for diagnostic verification of disease progression in cancer patients and selection of effective treatment options via development of the patient-derived xenograft (PDX) models for drug screening. The purpose of this study is to investigate the effects of cryoprotectant agents (CPAs) on the expression of BCL-2 family genes (BCL-2, BAX, and BAD) that are involved in the growth and development of breast cancers. MCF-7 cells were cryopreserved in Dulbecco's modified Eagle's medium (DMEM) with 20% (v/v) fetal bovine serum, using 10% (v/v) Me2SO (dimethyl sulfoxide, DMSO) or 7.5% (v/v) Me2SO with 100is-300 mM trehalose as cryoprotectant solutions. After storage at -80°C for 7 days, the cells were thawed for evaluation. The use of Me2SO and trehalose has affected cell survival, proliferation, apoptotic state, as well as BCL-2 family gene expression. The conventional 10% (v/v) Me2SO method yields ∼80% post-thaw cell survival and good cell proliferation, but it drastically alters the pattern of the BCL-2 family gene expression. The antiapoptotic gene BCL-2 is downregulated, whereas two proapoptotic genes BAX and BAD are upregulated. The partial substitution of Me2SO with 200 or 300 mM trehalose enhances cell proliferation of survived cells after cryopreservation. The presence of trehalose upregulates the expression of both the antiapoptotic gene BCL-2 and proapoptotic genes BAX and BAD. Cryopreservation could tip off the checkpoint of the apoptotic pathway regulated by the BCL-2 family members, and the effect may be protectant dependent. The findings of this study demonstrate the importance of paying attention to the potential change of gene expression and metabolic state of cancer cells after cryopreservation in an attempt to development of the PDX models from cryopreserved cancer cells or tissue specimens.

Protection by Glycerol, Sucrose and Trehalose for Acellular Human Dermis Against Gamma Irradiation Damage at -78.5°C.

BACKGROUND: Glycerol, sucrose and trehalose are used as protectants for membrane, protein, cell and tissue preservation. The undercooled state (glassy or rubbery) of their solutions may also offer protection for protein, cells and tissues against radiation damage upon sterilization. OBJECTIVE: The study aimed to examine the protective effects of glycerol, sucrose and trehalose on cryopreserved acellular human dermis against gamma irradiation damage. MATERIALS AND METHODS: Acellular human dermis was cryopreserved at -80°C in glycerol, sucrose and trehalose solutions or their combinations with a base citrate-phosphate buffer (pH 6.0). Cryopreserved acellular dermis was then subjected to 13 kGy gamma irradiation at -78.5°C, and radiation damage was assessed by histological evaluation. RESULTS: Freeze and thaw alone do not alter the structure of acellular dermis, but gamma irradiation at -78.5°C results in significant structural changes in acellular dermis, including the formation of large holes, the damage of collagen fibers and the loss of overall dermis tissue histology. The incorporation of glycerol, sucrose and trehalose into cryopreservation solutions reduces gamma irradiation-induced tissue structural damage considerably. When used alone, trehalose (0.5 M) provided better protection against gamma irradiation damage than did sucrose (0.5 M) and glycerol (1.0 M). When used in combination, the glycerol and trehalose combination provides the best tissue protection. Significant donor-to-donor variation exists in tissue damage after gamma irradiation. For donor dermis that is less sensitive to gamma irradiation damage, glycerol, sucrose or trehalose alone is able to provide good protection. However, for more sensitive donor dermis, only the glycerol and trehalose combination is able to provide sufficient tissue protection. CONCLUSION: Glycerol, sucrose and trehalose protects cryopreserved acellular human dermis against gamma irradiation damage. Cryopreservation solutions can be optimized to permit tissues for gamma sterilization to increase the safety human tissue implants.

Improvement of calcium phosphate scaffold osteogenesis in vitro via combination of glutamate-modified BMP-2 peptides.

Alpha-tricalcium phosphate (α-TCP) based porous scaffolds have superior osteoconduction and osteoinduction in bone tissue engineering, furthermore, these 3D porous scaffolds can be used as efficient drug delivery carriers. In the concept of tissue engineering, the "drugs" could be defined as drug molecules or biomacromolecules, even cells. These "drugs" have endowed the scaffolds which were laden improved abilities compared with the blank scaffolds. In this study, we anchored osteogenic bone morphogenetic protein-2 (BMP-2) derived peptides to α-TCP 3D porous scaffolds by linking the E7 domain to the target peptides, constructed the modified active peptides (E7BMP-2 peptides) delivery system, which finally achieved the modified peptides sustaining release and enhanced rat bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation in vitro. The α-TCP 3D porous scaffolds had micropores and interconnected micropores which expanded surface area of the scaffolds. The release test testified the constructed the delivery system had realized long-term release in which the peptides dosage could be detected by the BCA protein assay kit after 10 days compared with BMP-2 proteins which absorbed on the same α-TCP 3D porous scaffolds. The constructed E7BMP-2 peptides delivery system supported rat BMSCs osteogenic differentiation in the form of improving the genes expression levels of Runx2, ALP and OCN. Based on electrostatic interactions, E7 domain fastened combination between the active BMP-2 derived peptides and the α-TCP 3D porous scaffolds, the sustaining E7BMP-2 peptides release promoted the BMSCs osteogenesis as BMP-2 proteins did, which endowed α-TCP 3D porous scaffolds enhanced osteoinductive abilities in vitro.

Quantification of α-Gal Antigen Removal in the Porcine Dermal Tissue by α-Galactosidase.

The α-Gal (Galα1,3-Galß1-4GlcNAc-R) epitope, the major xenoantigen, is the first barrier in a porcine-to-man tissue and organ xenotransplantation. The elimination or reduction of the α-Gal epitopes is therefore an important step for a successful xenotransplantation. The present study is to evaluate the α-Gal elimination in the porcine skin with α-galactosidase treatment, and to assess two methods (immunohistochemistry and inhibition ELISA) that may be used in quality control for quantifying the extent of the α-Gal elimination. Enzymatic cleavage in a single-step process is extremely efficient and affordable at eliminating the α-Gal epitope even in a tissue as dense as the porcine dermis. The cost of enzymatic cleavage is found to be less than US$7 for a 10 × 10 cm piece of porcine skin (0.5 mm thick) or about US$140 for 100 g of 3-dimensional soft tissues. After enzymatic cleavage, the α-Gal-positive immunostaining was essentially undetectable in enzyme-treated porcine skin. The inhibition rate constant of the monoclonal anti-Gal antibody M86 binding to α-Gal-bovine serum albumin in ELISA was reduced from 15.0 ± 4.3 (n = 10) to 6.1 ± 2.6 (n = 7) after enzyme treatment, in comparison to 4.4 ± 1.8 (n = 9) background inhibition of decellularized human skin (the ultimate negative control), which demonstrates ∼ 84% elimination of α-Gal epitopes in treated porcine skin. To examine the suitability of two detection methods for the routine quality control application, comparative studies were made with control and enzyme-treated porcine skin, porcine skin from the α-Gal knockout animal, as well as decellularized human skin. The data show that the traditional immunohistochemistry and, to a less extent, the inhibition ELISA with further modifications can be used as quality control tools in the production and selection of biocompatible bioprosthetic devices. The biological evaluation of enzyme-treated porcine skin is ongoing with a small animal model and a nonhuman primate model.

Calorimetric analysis of cryopreservation and freeze-drying formulations.

Differential scanning calorimetry (DSC) is a commonly used thermal analysis technique in cryopreservation and freeze-drying research. It has been used to investigate crystallization, eutectic formation, glass transition, devitrification, recrystallization, melting, polymorphism, molecular relaxation, phase separation, water transport, thermochemistry, and kinetics of complex reactions (e.g., protein denaturation). Such information can be used for the optimization of protective formulations and process protocols. This chapter gives an introduction to beginners who are less familiar with this technique. It covers the instrument and its basic principles, followed by a discussion of the methods as well as examples of specific applications.

Preclinical evaluations of acellular biological conduits for peripheral nerve regeneration.

Various types of natural biological conduits have been investigated as alternatives to the current surgical standard approach for peripheral nerve injuries. Autologous nerve graft, the current gold standard for peripheral nerve damage, is limited by clinical challenges such as donor-site morbidity and limited availability. The purpose of this study was to evaluate the efficacy of using acellular xenographic conduits (nerve, artery, and dermis) for the repair of a 1.2 cm critical size defect of peripheral nerve in a rodent model. Four months post surgery, the animal group receiving acellular artery as a nerve conduit showed excellent physiological outcome in terms of the prevention of muscle atrophy and foot ulcer. Histological assessment of the bridged site revealed excellent axon regeneration, as opposed to the nonrepaired control group or the group receiving dermal conduit. Finally, the study evaluated the potential improvement via the addition of undifferentiated mesenchymal stem cells into the artery conduit during the bridging procedure. The mesenchymal stem cell-dosed artery conduit group resulted in significantly higher concentration of regenerated axons over artery conduit alone, and exhibited accelerated muscle atrophy rescue. Our results demonstrated that xenographic artery conduits promoted excellent axonal regeneration with highly promising clinical relevance.

Process-induced extracellular matrix alterations affect the mechanisms of soft tissue repair and regeneration.

Extracellular matrices derived from animal tissues for human tissue repairs are processed by various methods of physical, chemical, or enzymatic decellularization, viral inactivation, and terminal sterilization. The mechanisms of action in tissue repair vary among bioscaffolds and are suggested to be associated with process-induced extracellular matrix modifications. We compared three non-cross-linked, commercially available extracellular matrix scaffolds (Strattice, Veritas, and XenMatrix), and correlated extracellular matrix alterations to in vivo biological responses upon implantation in non-human primates. Structural evaluation showed significant differences in retaining native tissue extracellular matrix histology and ultrastructural features among bioscaffolds. Tissue processing may cause both the condensation of collagen fibers and fragmentation or separation of collagen bundles. Calorimetric analysis showed significant differences in the stability of bioscaffolds. The intrinsic denaturation temperature was measured to be 51°C, 38°C, and 44°C for Strattice, Veritas, and XenMatrix, respectively, demonstrating more extracellular matrix modifications in the Veritas and XenMatrix scaffolds. Consequently, the susceptibility to collagenase degradation was increased in Veritas and XenMatrix when compared to their respective source tissues. Using a non-human primate model, three bioscaffolds were found to elicit different biological responses, have distinct mechanisms of action, and yield various outcomes of tissue repair. Strattice permitted cell repopulation and was remodeled over 6 months. Veritas was unstable at body temperature, resulting in rapid absorption with moderate inflammation. XenMatrix caused severe inflammation and sustained immune reactions. This study demonstrates that extracellular matrix alterations significantly affect biological responses in soft tissue repair and regeneration. The data offer useful insights into the rational design of extracellular matrix products and bioscaffolds of tissue engineering.

Aging of a regenerative biologic scaffold (AlloDerm native tissue matrix) during storage at elevated humidity and temperature.

Tissue products will age upon storage. Although a number of tissue products have been used for tissue repair and regeneration for more than a decade, no study has been published on the aging of tissue products and its potential effect on product function. This study investigated aging-caused changes in a regenerative biologic scaffold (AlloDerm native tissue matrix) upon storage at accelerated conditions. Tissue matrix was stored at elevated humidity (33%, 75%, and 85% relative humidity [RH]) and temperature (40 +/- 2 degrees C). The study measured the accumulation of advanced glycation end-products (Maillard products), and the changes of tissue structure, tissue stability, and mechanical properties as well as in vitro fibroblast repopulation. Tissue products stored at 75% RH and 85% RH changed significantly, including collagen condensation and cross-linking, increased breaking strength, and decreased elasticity. The aged products became less stable, as demonstrated by lower denaturation temperature, lower denaturation enthalpy, and higher susceptibility to nonspecific proteolytic action. In comparison, changes were nondetectable in control products stored at 2 degrees C to 8 degrees C, or very small in tissue products stored at 33% RH and 40 degrees C. Changes of tissue structure and stability were correlated highly with the formation of Maillard products, suggesting a role of Maillard reactions in tissue aging during storage. Calorimetric analysis revealed that tissue products stored at 2 degrees C to 8 degrees C and at 33% RH and 40 degrees C were in the glassy state, whereas the products stored at 75% RH, 85% RH, and 40 degrees C were not in the glassy state, suggesting a role of the glassy state in preserving tissue products during storage. Aging did not affect in vitro fibroblast proliferation on tissue matrix, and further tests are needed to investigate how aging may affect in vivo performance of the tissue product.