Development of a Static Avascular and Dynamic Vascular Human Skin Equivalent Employing Collagen/Keratin Hydrogels.

One of the primary complications in generating physiologically representative skin tissue is the inability to integrate vasculature into the system, which has been shown to promote the proliferation of basal keratinocytes and consequent keratinocyte differentiation, and is necessary for mimicking representative barrier function in the skin and physiological transport properties. We created a 3D vascularized human skin equivalent (VHSE) with a dermal and epidermal layer, and compared keratinocyte differentiation (immunomarker staining), epidermal thickness (H&E staining), and barrier function (transepithelial electrical resistance (TEER) and dextran permeability) to a static, organotypic avascular HSE (AHSE). The VHSE had a significantly thicker epidermal layer and increased resistance, both an indication of increased barrier function, compared to the AHSE. The inclusion of keratin in our collagen hydrogel extracellular matrix (ECM) increased keratinocyte differentiation and barrier function, indicated by greater resistance and decreased permeability. Surprisingly, however, endothelial cells grown in a collagen/keratin extracellular environment showed increased cell growth and decreased vascular permeability, indicating a more confluent and tighter vessel compared to those grown in a pure collagen environment. The development of a novel VHSE, which incorporated physiological vasculature and a unique collagen/keratin ECM, improved barrier function, vessel development, and skin structure compared to a static AHSE model.

Keratose Hydrogel Drives Differentiation of Cardiac Vascular Smooth Muscle Progenitor Cells: Implications in Ischemic Treatment.

Peripheral artery disease (PAD) is a common vascular disorder in the extremity of limbs with limited clinical treatments. Stem cells hold great promise for the treatment of PAD, but their therapeutic efficiency is limited due to multiple factors, such as poor engraftment and non-optimal selection of cell type. To date, stem cells from a variety of tissue sources have been tested, but little information is available regarding vascular smooth muscle cells (VSMCs) for PAD therapy. The present study examines the effects of keratose (KOS) hydrogels on c-kit+/CD31- cardiac vascular smooth muscle progenitor cell (cVSMPC) differentiation and the therapeutic potential of the resultant VSMCs in a mouse hindlimb ischemic model of PAD. The results demonstrated that KOS but not collagen hydrogel was able to drive the majority of cVSMPCs into functional VSMCs in a defined Knockout serum replacement (SR) medium in the absence of differentiation inducers. This effect could be inhibited by TGF-ß1 antagonists. Further, KOS hydrogel increased expression of TGF-ß1-associated proteins and modulated the level of free TGF-ß1 during differentiation. Finally, transplantation of KOS-driven VSMCs significantly increased blood flow and vascular densities of ischemic hindlimbs. These findings indicate that TGF-ß1 signaling is involved in KOS hydrogel-preferred VSMC differentiation and that enhanced blood flow are likely resulted from angiogenesis and/or arteriogenesis induced by transplanted VSMCs.

Keratin Promotes Differentiation of Keratinocytes Seeded on Collagen/Keratin Hydrogels.

Keratinocytes undergo a complex process of differentiation to form the stratified stratum corneum layer of the skin. In most biomimetic skin models, a 3D hydrogel fabricated out of collagen type I is used to mimic human skin. However, native skin also contains keratin, which makes up 90% of the epidermis and is produced by the keratinocytes present. We hypothesized that the addition of keratin (KTN) in our collagen hydrogel may aid in the process of keratinocyte differentiation compared to a pure collagen hydrogel. Keratinocytes were seeded on top of a 100% collagen or 50/50 C/KTN hydrogel cultured in either calcium-free (Ca-free) or calcium+ (Ca+) media. Our study demonstrates that the addition of keratin and calcium in the media increased lysosomal activity by measuring the glucocerebrosidase (GBA) activity and lysosomal distribution length, an indication of greater keratinocyte differentiation. We also found that the presence of KTN in the hydrogel also increased the expression of involucrin, a differentiation marker, compared to a pure collagen hydrogel. We demonstrate that a combination (i.e., containing both collagen and kerateine or "C/KTN") hydrogel was able to increase keratinocyte differentiation compared to a pure collagen hydrogel, and the addition of calcium further increased the differentiation of keratinocytes. This multi-protein hydrogel shows promise in future models or treatments to increase keratinocyte differentiation into the stratum corneum.

Keratose hydrogel for tissue regeneration and drug delivery.

Keratin (KRT), a natural fibrous structural protein, can be classified into two categories: "soft" cytosolic KRT that is primarily found in the epithelia tissues (e.g., skin, the inner lining of digestive tract) and "hard" KRT that is mainly found in the protective tissues (e.g., hair, horn). The latter is the predominant form of KRT widely used in biomedical research. The oxidized form of extracted KRT is exclusively denoted as keratose (KOS) while the reduced form of KRT is termed as kerateine (KRTN). KOS can be processed into various forms (e.g., hydrogel, films, fibers, and coatings) for different biomedical applications. KRT/KOS offers numerous advantages over other types of biomaterials, such as bioactivity, biocompatibility, degradability, immune/inflammatory privileges, mechanical resilience, chemical manipulability, and easy accessibility. As a result, KRT/KOS has attracted considerable attention and led to a large number of publications associated with this biomaterial over the past few decades; however, most (if not all) of the published review articles focus on KRT regarding its molecular structure, biochemical/biophysical properties, bioactivity, biocompatibility, drug/cell delivery, and in vivo transplantation, as well as its applications in biotechnical products and medical devices. Current progress that is directly associated with KOS applications in tissue regeneration and drug delivery appears an important topic that merits a commentary. To this end, the present review aims to summarize the current progress of KOS-associated biomedical applications, especially focusing on the in vitro and in vivo effects of KOS hydrogel on cultured cells and tissue regeneration following skin injury, skeletal muscle loss, peripheral nerve injury, and cardiac infarction.

Keratin Biomaterials Improve Functional Recovery in a Rat Spinal Cord Injury Model.

STUDY DESIGN: Laboratory study using a rat T9 contusion model of spinal cord injury (SCI). OBJECTIVE: The purpose of this study was to evaluate which method of delivery of soluble keratin biomaterials would best support functional restoration through the macrophage polarization paradigm. SUMMARY OF BACKGROUND DATA: SCI is a devastating neurologic event with complex pathophysiological mechanisms that currently has no cure. After injury, macrophages and resident microglia are key regulators of inflammation and tissue repair exhibiting phenotypic and functional plasticity. Keratin biomaterials have been demonstrated to influence macrophage polarization and promote the M2 anti-inflammatory phenotype that attenuates inflammatory responses. METHODS: Anesthetized female Lewis rats were subjected to moderate T9 contusion SCI and randomly divided into: no therapy (control group), an intrathecally injected keratin group, and a keratin-soaked sponge group (n = 11 in all groups). Functional recovery assessments were obtained at 3- and 6-weeks post-injury (WPI) using gait analysis performed with the DigiGait Imaging System treadmill and at 1, 3, 7, 14, 21, 28, 35, and 42 days post-injury by the Basso, Beattie, Bresnahan (BBB) locomotor rating scale. Histology and immunohistochemistry of serial spinal cord sections were performed to assess injury severity and treatment efficacy. RESULTS: Compared to control rats, applying keratin materials after injury improved functional recovery in certain gait parameters and overall trended toward significance in BBB scores; however, no significant differences were observed with tissue analysis between groups at 6 WPI. CONCLUSION: Results suggest that keratin biomaterials support some locomotor functional recovery and may alter the acute inflammatory response by inducing macrophage polarization following SCI. This therapy warrants further investigation into treatment of SCI.Level of Evidence: N/A.

Collagen/kerateine multi-protein hydrogels as a thermally stable extracellular matrix for 3D in vitro models.

Objective: To determine whether the addition of kerateine (reduced keratin) in rat tail collagen type I hydrogels increases thermal stability and changes material properties and supports cell growth for use in cellular hyperthermia studies for tumor treatment.Methods: Collagen type I extracted from rat tail tendon was combined with kerateine extracted from human hair fibers. Thermal, mechanical, and biocompatibility properties and cell behavior was assessed and compared to 100% collagen type I hydrogels to demonstrate their utility as a tissue model for 3D in vitro testing.Results: A combination (i.e., containing both collagen 'C/KNT') hydrogel was more thermally stable than pure collagen hydrogels and resisted thermal degradation when incubated at a hyperthermic temperature of 47°C for heating durations up to 60 min with a higher melting temperature measured by DSC. An increase in the storage modulus was only observed with an increased collagen concentration rather than an increased KTN concentration; however, a change in ECM structure was observed with greater fiber alignment and width with an increase in KTN concentration. The C/KTN hydrogels, specifically 50/50 C/KTN hydrogels, also supported the growth and of fibroblasts and MDA-MB-231 breast cancer cells similar to those seeded in 100% collagen hydrogels.Conclusion: This multi-protein C/KTN hydrogel shows promise for future studies involving thermal stress studies without compromising the 3D ECM environment or cell growth.

Comprehensive characterization of the human pancreatic proteome for bioengineering applications.

Interactions between the pancreatic extracellular matrix (ECM) and islet cells are known to regulate multiple aspects of islet physiology, including survival, proliferation, and glucose-stimulated insulin secretion. Recognizing the essential role of ECM in islet survival and function, various engineering approaches have been developed that aim to utilize ECM-based materials to recreate a native-like microenvironment. However, a major impediment to the success of these approaches has been the lack of a robust and comprehensive characterization of the human pancreatic proteome. Herein, by combining mass spectrometry (MS) and multiplex ELISA, we have provided an improved workflow for the in-depth profiling of the proteome, including minor constituents that are generally underrepresented. Moreover, we have further validated the effectiveness of our detergent-free decellularization protocol in the removal of cellular proteins and retention of the matrisome. It has also been established that the decellularized ECM and its derivatives can provide more tissue-specific cues than traditionally used biological scaffolds and are therefore more physiologically relevant for the development of hydrogels, bioinks and medium additives, in order to create a pancreatic niche. The data generated in this study would contribute significantly to the efforts of comprehensively defining the ECM atlas and also serve as a standard for the human pancreatic proteome to provide further guidance for design and engineering strategies for improved tissue engineering scaffolds.

Application of the coal mine floor rating (CMFR) to assess the floor stability in a Central Appalachian Coal Mine.

Estimating the overall floor stability in a coal mine using deterministic methods which require complex engineering properties of floor strata is desirable, but generally it is impractical due to the difficulty of gathering essential input data. However, applying a quantitative methodology to describe floor quality with a single number provides a practical estimate for preliminary assessment of floor stability. The coal mine floor rating (CMFR) system, developed by the University of New South Wales (UNSW), is a rock-mass classification system that provides an indicator for the competence of floor strata. The most significant components of the CMFR are uniaxial compressive strength and discontinuity intensity of floor strata. In addition to the competence of the floor, depth of cover and stress notch angle are input parameters used to assess the preliminary floor stability. In this study, CMFR methodology was applied to a Central Appalachian Coal Mine that intermittently experienced floor heave. Exploratory drill core data, overburden maps, and mine plans were utilized for the study. Additionally, qualitative data (failure/non-failure) on floor conditions of the mine entries near the core holes was collected and analyzed so that the floor quality and its relation to entry stability could be estimated by statistical methods. It was found that the current CMFR classification system is not directly applicable in assessing the floor stability of the Central Appalachian Coal Mine. In order to extend the applicability of the CMFR classification system, the methodology was modified. A calculation procedure of one of the CMFR classification system's components, the horizontal stress rating (HSR), was changed and new parameters were added to the HSR.

Coal mine entry rating system: A case study.

Coal mines are continuously seeking to determine the performance of entries with different ground control products and installation methods. There are many factors that impact how an entry will perform which include but are not limited to geology, overburden, bolting type and pattern, and mine design. At the National Institute for Occupational Safety and Health (NIOSH), research has been instituted to examine the relationship of the parts of a coal mine entry as a system and not as individual components. To study this relationship, the first step in this study was to create a numeric rating system that accurately reflects visual observations of the mine entry and is easy to implement. NIOSH researchers devised this rating system to improve upon previous ideas, offering increased flexibility which can be incorporated into an overall entry condition that offers different levels of confidence based on the user's time devoted to the inspection. This new entry rating system was implemented at three different mines over varying periods of time to evaluate the ground response to the geology, bolt installation pattern, stress changes by mining, overburden, and time dependency.

Moderate Cover Bleeder Entry and Standing Support Performance in a Longwall Mine: a Case Study.

Bleeder entries are critically important to longwall mining for the moving of supplies, personnel, and the dilution of mine air contaminants. By design, these entries must stay open for many years for ventilation. Standing supports in moderate cover bleeder entries were observed, numerically modeled, and instrumented by researchers at the National Institute for Occupational Safety and Health (NIOSH). The measurements of the installed borehole pressure cells (BPCs), standing support load cells and convergence meters, and roof extensometers are presented in this paper in addition to the numerical modeling results and visual observations made by the NIOSH researchers in the bleeder entries. The results include the effects of multiple panels being extracted in close proximity to the instrumented site as well as over one and a half years of aging. As expected, standing supports closer to the longwall gob showed the greatest load and convergence. The roof sag appeared generally independent of the proximity to the longwall gob. The BPC readings were driven by both the proximity to the gob and the depth into the pillar. The results of this study demonstrated that the entry roof can respond independently of the pillar and standing support loading. In addition, the rear abutment stress experienced by this bleeder entry design was minimal. The closer the mine development, pillar, or supports are to the gob, the greater the applied load due to rear abutment stress.

A keratin-based microparticle for cell delivery.

Keratin-based biomaterials represent an attractive opportunity in the fields of wound healing and tissue regeneration, not only for their chemical and physical properties, but also for their ability to act as a delivery system for a variety of payloads. Importantly, keratins are the only natural biomaterial that is not targeted by specific tissue turnover-related enzymes, giving it potential stability advantages and greater control over degradation after implantation. However, in-situ polymerization chemistry in some keratin systems are not compatible with cells, and incorporation within constructs such as hydrogels may lead to hypoxia and cell death. To address these challenges, we envisioned a pre-formed keratin microparticle on which cells could be seeded, while other payloads (e.g. drugs, growth factors or other biologic compounds) could be contained within, although studies investigating the potential partitioning between phases during emulsion polymerization would need to be conducted. This study employs well-established water-in-oil emulsion procedures as well as a suspension culture method to load keratin-based microparticles with bone marrow-derived mesenchymal stem cells. Fabricated microparticles were characterized for size, porosity and surface structure and further analyzed to investigate their ability to form gels upon hydration. The suspension culture technique was validated based on the ability for loaded cells to maintain their viability and express actin and vinculin proteins, which are key indicators of cell attachment and growth. Maintenance of expression of markers associated with cell plasticity was also investigated. As a comparative model, a collagen-coated microparticle (Sigma) of similar size was used. Results showed that an oxidized form of keratin ("keratose" or "KOS") formed unique microparticle structures of various size that appeared to contain a fibrous sub-structure. Cell adhesion and viability was greater on keratin microparticles compared to collagen-coated microparticles, while marker expression was retained on both.

Assessing support alternatives for longwall gateroads subject to changing stress.

Longwall gateroad entries are subject to changing horizontal and vertical stress induced by redistribution of loads around the extracted panel. The stress changes can result in significant deformation of the entries that may include roof sag, rib dilation, and floor heave. Mine operators install different types of supports to control the ground response and maintain safe access and ventilation of the longwall face. This paper describes recent research aimed at quantifying the effect of longwall-induced stress changes on ground stability and using the information to assess support alternatives. The research included monitoring of ground and support interaction at several operating longwall mines in the U.S., analysis and calibration of numerical models that adequately represent the bedded rock mass, and observation of the support systems and their response to changes in stress. The models were then used to investigate the impact of geology and stress conditions on ground deformation and support response for various depths of cover and geologic scenarios. The research results were summarized in two regression equations that can be used to estimate the likely roof deformation and height of roof yield due to longwall-induced stress changes. This information is then used to assess the ability of support systems to maintain the stability of the roof. The application of the method is demonstrated with a retrospective analysis of the support performance at an operating longwall mine that experienced a headgate roof fall. The method is shown to produce realistic estimates of gateroad entry stability and support performance, allowing alternative support systems to be assessed during the design and planning stage of longwall operations.

Assessing Longwall Gateroad Ground Response and Support Alternatives.

Ground falls in longwall gateroad entries remain a concern in modern longwall operations. The gateroads are subject to changing horizontal and vertical ground stress induced by longwall extraction. These stress changes can result in failure of the strata around an entry leading to large deformations of the entry roof, floor, and ribs. The gateroad support systems are required to control the failed strata while maintaining safe access to the longwall face and unimpeded ventilation. This paper presents research that was conducted to better understand the stability issues in gateroad excavations and to develop procedures for evaluating support and layout alternatives for longwall gateroads. Using the results of a field-monitoring program and numerical model analysis of case histories, a conceptual model of gateroad support needs was developed. The conceptual model formed the basis for developing a set of equations that can be used to estimate likely roof sag and support loading for given roof geology and longwall-induced loading conditions. The developed equations were used to compare predicted gateroad stability to field study results, showing satisfactory agreement. The calculation procedures are used to demonstrate their application in assessing support alternatives at a case study mine. It is concluded that the developed analysis procedures provide realistic assessments of likely ground stability and can be used to evaluate alternative gateroad support systems at operating longwall mines.

Geologic data collection and assessment techniques in coal mining for ground control.

The identification and mitigation of adverse geologic conditions are critical to the safety and productivity of underground coal mining operations. To anticipate and mitigate adverse geologic conditions, a formal method to evaluate geotechnical factors must be established. Each mine is unique and has its own separate approach for defining what an adverse geological condition consists of. The collection of geologic data is a first critical step to creating a geological database to map these hazards efficiently and effectively. Many considerations must be taken into account, such as lithology of immediate roof and floor strata, seam height, gas and oil wells, faults, depressions in the mine floor (water) and increases in floor elevation (gas), overburden, streams and horizontal stress directions, amongst many other factors. Once geologic data is collected, it can be refined and integrated into a database that can be used to develop maps showing the trend, orientation, and extent of the adverse geological conditions. This information, delivered in a timely manner, allows mining personnel to be proactive in mine planning and support implementations, ultimately reducing the impacts of these features. This paper covers geologic exploratory methods, data organization, and the value of collecting and interpreting geologic information in coal mines to enhance safety and production. The implementation of the methods described above has been proven effective in predicting and mitigating adverse geologic conditions in underground coal mining. Consistent re-evaluation of data collection methods, geologic interpretations, mapping procedures, and communication techniques ensures continuous improvement in the accuracy of predictions and mitigation of adverse geologic conditions. Providing a concise record of the work previously done to track geologic conditions at a mine will allow for the smoothest transition during employee turnover and transitions. With refinements and standardization of data collection methods, such as those described in this paper, along with improvement in technology, the evaluation of adverse geologic conditions will evolve and continue to improve the safety and productivity of underground coal mining.

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production.

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

A comparative study of materials assembled from recombinant K31 and K81 and extracted human hair keratins.

Natural biopolymers have found success in tissue engineering and regenerative medicine applications. Their intrinsic biocompatibility and biological activity make them well suited for biomaterials development. Specifically, keratin-based biomaterials have demonstrated utility in regenerative medicine applications including bone regeneration, wound healing, and nerve regeneration. However, studies of structure-function relationships in keratin biomaterials have been hindered by the lack of homogeneous preparations of materials extracted and isolated from natural sources such as wool and hair fibers. Here we present a side-by-side comparison of natural and recombinant human hair keratin proteins K31 and K81. When combined, the recombinant proteins (i.e. rhK31 and rhK81) assemble into characteristic intermediate filament-like fibers. Coatings made from natural and recombinant dimers were compared side-by-side and investigated for coating characteristics and cell adhesion. In comparison to control substrates, the recombinant keratin materials show a higher propensity for inducing involucrin and hence, maturation in terms of potential skin cell differentiation.

Success and efficiency of cell seeding in Avian Tendon Xenografts - A promising alternative for tendon and ligament reconstruction.

Decellularized tendon xenografts offer a promising alternative for reconstruction by using ubiquitously available material. This study compares static and centrifugal seeding of avian tendon scaffolds with NIH 3T3 fibroblasts. Incorporation of viable cells was achievable with both techniques, represented by DNA content. Proliferation rate and viability assay showed neither damage by centrifugal force nor superiority of the technique. Cell proliferation after 10 days of culture demonstrated that the scaffold did not hinder 3-D culturing. Confocal laser microscopy revealed structural details as formation of focal adhesions, to provide deeper insight into the process of cell attachment and growth in xenografts.

Development and characterization of a biomimetic coating for percutaneous devices.

Percutaneous osseointegrated prosthetics (POP), which consist of a metallic post attached to the bone that extends outward through the skin to connect to an external prosthesis, have become a clinically relevant option to replace the typical socket-residual limb connection. POP devices offer several advantages such as mechanical off-loading of soft tissues, direct force transfer to the musculoskeletal system, greater proprioception, and overall improvement in limb kinesis compared to a socket system. However, POP devices create several challenges including epidermal downgrowth, increased infection risk, and mechanical tearing at the skin-implant interface. To address these issues, biomimetic surfaces and coatings have been developed in an attempt to create an infection-free and cohesive interface between POP devices and skin. The fingernail is a prime example of a natural system with a skin interface that is both mechanically and biologically stable. Exploiting keratins' previously demonstrated tissue compatibility and creating a biomimetic coating for POP devices that can imitate the human fingernail, and demonstrating its ability to promote a stable interface with skin tissue is the goal of this work. Silane coupling aided in producing a coating on titanium substrates consisting of human keratin proteins. Several combinations of silane and keratin derivatives were investigated, and in general showed a nano-scale coating thickness that supported skin cell (i.e. fibroblast and keratinocyte) adhesion. Initial enzyme-mediated degradation resistance was also demonstrated, but the coatings appeared to degrade at long time periods. Importantly, keratinocytes showed a stable phenotype with no indication of wound healing-like activity. These data provide justification to further explore keratin biomaterials for POP coatings that may stabilize the implant-skin interface.

A porcine xenograft-derived bone scaffold is a biocompatible bone graft substitute: An assessment of cytocompatibility and the alpha-Gal epitope.

BACKGROUND: Xenografts are an attractive alternative to traditional bone grafts because of the large supply from donors with predictable morphology and biology as well as minimal risk of human disease transmission. Clinical series involving xenograft bone transplantation, most commonly from bovine sources, have reported poor results with frequent graft rejection and failure to integrate with host tissue. Failures have been attributed to residual alpha-Gal epitope in the xenograft which humans produce natural antibody against. To the authors' knowledge, there is currently no xenograft-derived bone graft substitute that has been adopted by orthopedic surgeons for routine clinical use. METHODS: In the current study, a bone scaffold intended to serve as a bone graft substitute was derived from porcine cancellous bone using a tissue decellularization and chemical oxidation protocol. In vitro cytocompatibility, pathogen clearance, and alpha-Gal quantification tests were used to assess the safety of the bone scaffold intended for human use. RESULTS: In vitro studies showed the scaffold was free of processing chemicals and biocompatible with mouse and human cell lines. When bacterial and viral pathogens were purposefully added to porcine donor tissue, processing successfully removed these pathogens to comply with sterility assurance levels established by allograft tissue providers. Critically, 98.5% of the alpha-Gal epitope was removed from donor tissue after decellularization as shown by ELISA inhibition assay and immunohistochemical staining. CONCLUSIONS: The current investigation supports the biologic safety of bone scaffolds derived from porcine donors using a decellularization protocol that meets current sterility assurance standards. The majority of the highly immunogenic xenograft carbohydrate was removed from donor tissue, and these findings support further in vivo investigation of xenograft-derived bone tissue for orthopedic clinical application.