A Feasibility and Safety Study of a Novel Human Decellularized Dermal Matrix to Accelerate Healing of Neuropathic Diabetic Foot Ulcers in People With Type 1 and Type 2 Diabetes.

OBJECTIVES: The purpose of this study was to determine the feasibility and safety of a novel decellularized dermal matrix (DDM) for the treatment of chronic diabetic foot ulcers (DFUs). METHODS: An interventional, single-arm, prospective study of DDM for DFU treatment was conducted in 2 Canadian centres from July 1, 2016 to May 30, 2017. Individuals ≥18 years of age, with an active DFU of ≥2 weeks and ulcer area ≥1 cm2 before debridement and who consented to participate, were enrolled in this clinical trial. RESULTS: A total of 11 patients were enrolled, with 9 patients (82%) having achieved 100% closure between 2 and 8 weeks. The mean and median times to wound closure for these patients were 3.3 and 2.5 weeks, respectively. The mean and median reductions in wound area at 4 weeks posttreatment were 87% and 100%, respectively. The proportion of patients having achieved complete healing at 12 weeks was 82%. All patients received only 1 DDM application to achieve these results. There were no adverse events related to use of the product. No cases of recurrence during a 1-year follow up after completion of the study were reported for patients who achieved wound closure. CONCLUSIONS: These findings provide evidence that this DDM may be safe and effective for the treatment of chronic, hard-to-heal neuropathic DFUs. Specifically, DDM demonstrated the potential to accelerate healing of DFUs when compared with reported times of 8 to 12 weeks required to achieve closure using the current standard of care.

Precision cell delivery in biphasic polymer systems enhances growth of keratinocytes in culture and promotes their attachment on acellular dermal matrices.

Current approaches for precision deposition of cells are not optimized for moist environments or for substrates with complex surface topographic features, for example, the surface of dermal matrices and other biomaterials. To overcome these challenges, an approach is presented that utilizes cell confinement in phase-separating polymer solutions of polyethylene glycol and dextran to precisely deliver keratinocytes in well-defined colonies. Using this approach, keratinocyte colonies are produced with superior viability, proliferative capacity, and barrier formation compared with the same number of cells dispersedly seeded across substrate surfaces. It is further demonstrated that keratinocytes delivered in colonies to the surface of acellular dermal matrices form an intact epidermal basal layer more rapidly and more completely than cells delivered by conventional dispersed seeding. These findings demonstrate that delivery of keratinocytes in phase-separating polymer solutions holds potential for enhancing growth of keratinocytes in culture and production of functional skin equivalents.

Efficient decellularization of rabbit trachea to generate a tissue engineering scaffold biomatrix.

OBJECTIVES: Most tracheal decellularization protocols are lengthy and can lead to reduced biomechanical stability. The objectives of this study were: 1) to generate a tracheal extracellular matrix scaffold using an efficient decellularization process and 2) to characterize the decellularized scaffold to assess its suitability for tissue engineering applications. METHODS: Twelve rabbit tracheae underwent a decellularization process that involved enzymatic-detergent treatments. For characterization, fresh (control) and decellularized tissues underwent histological, immunohistochemical, and biochemical analyses. Tensile testing, scanning electron microscopy, and biocompatibility assay were also conducted. RESULTS: Post-decellularization, the tracheal tissue had significantly less genetic material while the structural integrity was maintained. Specifically, the deoxyribonucleic acid content was significantly reduced and the glycosaminoglycan content was unchanged. Cell and cellular components were largely removed; at the same time the tensile properties and surface ultrastructural characteristics were unaltered. Biocompatibility was confirmed by contact cytotoxicity assay. CONCLUSIONS: Overall, an efficient decellularization process was used to treat rabbit tracheal tissue. The effectiveness of the decellularization process was demonstrated and at the same time there was preservation of the underlying extracellular matrix structure. This decellularized material may serve as a potential scaffold for tracheal tissue engineering.

Development and characterization of decellularized human nasoseptal cartilage matrix for use in tissue engineering.

OBJECTIVES/HYPOTHESIS: Reconstruction of cartilage defects in the head and neck can require harvesting of autologous cartilage grafts, which can be associated with donor site morbidity. To overcome this limitation, tissue-engineering approaches may be used to generate cartilage grafts. The objective of this study was to decellularize and characterize human nasoseptal cartilage with the aim of generating a biological scaffold for cartilage tissue engineering. STUDY DESIGN: Laboratory study using nasoseptal cartilage. METHODS: Remnant human nasoseptal cartilage specimens were collected and subjected to a novel decellularization treatment. The decellularization process involved several cycles of enzymatic detergent treatments. For characterization, decellularized and fresh (control) specimens underwent histological, biochemical, and mechanical analyses. Scanning electron microscopy and biocompatibility assay were also performed. RESULTS: The decellularization process had minimal effect on glycosaminoglycan content of the cartilage extracellular matrix. Deoxyribonucleic acid (DNA) analysis revealed the near-complete removal of genomic DNA from decellularized tissues. The effectiveness of the decellularization process was also confirmed on histological and scanning electron microscopic analyses. Mechanical testing results showed that the structural integrity of the decellularized tissue was maintained, and biocompatibility was confirmed. CONCLUSION: Overall, the current decellularization treatment resulted in significant reduction of genetic/cellular material with preservation of the underlying extracellular matrix structure. This decellularized material may serve as a potential scaffold for cartilage tissue engineering. LEVEL OF EVIDENCE: N/A. Laryngoscope, 126:2226-2231, 2016.

Repair of tympanic membrane perforation using novel adjuvant therapies: a contemporary review of experimental and tissue engineering studies.

OBJECTIVE: To perform a contemporary review of experimental studies to describe the effects of various novel adjuvant therapies in enhancing tympanic membrane (TM) perforation healing. METHODS: A PubMed search for articles from January 2000 to June 2012 related to TM perforation, along with the references of those articles, was performed. Inclusion and exclusion criteria were applied to all experimental studies assessing adjuvant therapies to TM healing. RESULTS: Many studies have assessed the efficacy of biomolecules or growth factors, such as epidermal growth factors and basic fibroblast growth factors, in TM regeneration with significant success. More recent strategies in TM tissue engineering have involved utilizing bioengineered scaffold materials, such as silk fibroin, chitosan, calcium alginate, and decellularized extracellular matrices. Most scaffold materials demonstrated biocompatibility and faster TM perforation healing rates. CONCLUSION: Although several studies have demonstrated promising results, many questions still remain, such as the adequacy of animal models and long-term biocompatibility of adjuvant materials. As well, further studies comparing various adjuvant substances and bioscaffolds are required prior to clinical application.

Effect of basic fibroblast growth factor on the cellular repopulation of decellularized anterior cruciate ligament allografts.

The use of decellularized anterior cruciate ligament (ACL) allografts in ACL replacement surgery may allow for the native structure of the ligament to be retained, thereby recapturing the function of the ligament post-injury. Our previous work has focused on repopulating decellularized allograft ACL tissue with ACL fibroblasts in order to prevent destructive remodelling of the implanted tissue by extrinsic host cells. In this study, the use of basic fibroblast growth factor (bFGF) to improve the cellular repopulation of decellularized ACL tissue was assessed. A concentration of 6 ng/ml bFGF was demonstrated to be effective in increasing cellular growth in the absence of tissue; however, this concentration, as well as reduced and increased levels of bFGF (0.1 and 60 ng/ml, respectively), failed to increase cellular repopulation of ACL fibroblast-seeded decellularized tissue after 28 days of culture. Mean repopulation levels of 11-19% of fresh tissue [3200-5300 cells/mg dry weight (dwt) tissue] were achieved after 28 days in culture. Qualitative observation of histological samples suggested that different repopulation characteristics exist at various concentrations of bFGF and, in particular, that bFGF may be stimulating a catabolic pathway resulting in matrix destruction. Significant differences in the effects of bFGF observed between cell-only and cell-and-tissue studies serve to reinforce the concept that cells respond to stimuli in a different manner, depending on the surrounding environment. As a result, caution should be used when information obtained from studies utilizing cells alone is applied to the development of tissue-engineered constructs.

The effect of chemical modification of amino acid side-chains on collagen degradation by enzymes.

In this study, the effects of specific chemical modifications of amino acid side-chains on the in vitro enzyme degradation of type I collagen was studied. Two monofunctional epoxides of different size and chemistry were used to modify lysine and methylglyoxal was used to modify arginine. Lysine residues were modified using glycidol, a small hydrophilic reagent or n-butylglycidylether, a larger hydrophobic reagent. Amino acid analysis, swelling measurements, in vitro enzyme degradation analyses (using either collagenase, trypsin, acetyltrypsin, or cathepsin B), and gel chromatography were used to determine the effects of each chemical modification on purified type I collagen. Collagen solubilization by enzymes depended upon the size and chemistry of epoxides used to modify lysine residues. Modification of lysine residues by glycidol and arginine modification by methylglyoxal together significantly reduced collagen solubilization by acetyltrypsin and collagenase, whereas increased collagen solubilization was observed for all enzymes after lysine modification with n-butylglycidylether combined with arginine modification by methylglyoxal. Gel chromatographic analyses of collagen fragments solubilized by acetyltrypsin from type I collagen revealed that both the extent of solubilization and sites of cleavage were altered after lysine and arginine modification. In contrast, lysine and arginine modification only altered the amount of collagen solubilized by collagenase and had no effect on the amount collagen solubilized by cathepsin B. The ability to modulate the enzyme degradation of collagen-based materials as demonstrated in this study may facilitate the design of novel scaffolds for tissue regeneration or collagen-based drug/protein/gene delivery systems.

Matrix alteration and not residual sodium dodecyl sulfate cytotoxicity affects the cellular repopulation of a decellularized matrix.

It has been suggested that residual cytotoxic sodium dodecyl sulfate (SDS) is responsible for the low levels of cell in-growth observed in SDS decellularized tissues. To determine whether this is the case, we used 2 washing methods to remove residual SDS and extensive biochemical, mechanical, and structural analyses to determine the effects of SDS-based decellularization on porcine anterior cruciate ligament (ACL) tissue and its propensity for cellular repopulation. The level of residual SDS in decellularized tissue was reduced using 2 different washing techniques (pH = 9 buffer, 75% ethanol). After washing in pH = 9 or 75% ethanol, residual SDS concentrations in decellularized tissues were found to be approximately 8 and 23 times less than reported SDS cytotoxic levels, respectively. It was found that SDS treatment significantly reduced glycosaminoglycan levels, increased collagen crimp amplitude and periodicity, and increased susceptibility of collagen to degradation by the gelatinase enzyme trypsin. The level of repopulation and viability of autologous ACL fibroblasts in the decellularized tissue after 28 days of culture were found to be the same regardless of the washing technique and resulting level of residual SDS in the tissue. This strongly indicates that alterations in tissue matrix biochemistry or structure from SDS treatment and not residual SDS cytotoxicity are responsible for the low cell re-population observed in SDS decellularized tissues.

Effect of extraction protocols and epidermal growth factor on the cellular repopulation of decellularized anterior cruciate ligament allografts.

We are developing a decellularized bone-anterior cruciate ligament (ACL)-bone allograft for treatment of ACL disruption in young or active patients. This study demonstrates the feasibility of seeding decellularized ACL tissue with primary ligament fibroblasts. Porcine ACLs were decellularized by one of three protocols, each differing only by the detergent/solvent used during the second wash (SDS, Triton-X, or TnBP). Porcine ACL fibroblasts were obtained by explant and seeded onto tissue samples of decellularized ACL. Culture conditions were varied to compare the relative effect of three different decellularization protocols on cellular repopulation. Culture condition variables included (1) the number of cells used for seeding, (2) the addition of epidermal growth factor (EGF), and (3) culture duration. Cellular ingrowth was assessed by metabolic activity (MTT assay), DNA quantification (Hoescht dye), and histology (H&E staining). Cell counting on histological sections demonstrated that Triton-X-and TnBP-treated ligaments were more receptive to cellular ingrowth than SDS-treated samples. The addition of EGF to culture medium did not significantly increase cellular ingrowth. Both the Triton-X and TnBP decellularization treatments provide suitable, naturally derived scaffolds for the ingrowth of primary ACL fibroblasts, and should be further investigated in the development of an allograft-derived bone-ACL-bone graft.

Effectiveness of three extraction techniques in the development of a decellularized bone-anterior cruciate ligament-bone graft.

In this study, porcine bone-anterior cruciate ligament-bone (B-ACL-B) grafts were decellularized using one of three protocols incorporating surfactants lauryl sulfate (SDS), Triton X-100, and/or an organic solvent (tributyl phosphate (TnBP)). The effectiveness of Triton-SDS, Triton-Triton or Triton-TnBP treatments in removing cellular materials was determined and possible changes in biochemical composition and mechanical properties due to each treatment were investigated. Treatment with Triton-SDS was most effective at removing cell nuclei and intracellular protein (vimentin) from the ACL but affected both the collagen and glycosaminoglycan (GAG) components of the extracellular matrix while increasing the tensile stiffness of the ligament. Triton-Triton was the least effective of the three treatments in terms of cellular extraction, but did not significantly change the mechanical and biochemical properties of the ACL. Triton-TnBP matched the level of decellularization achieved by Triton-SDS in terms of visible cell nuclei; however, the extraction of intracellular vimentin was less consistent. TnBP treatment also slightly decreased the collagen content of the ACL but did not alter its mechanical properties. Overall, all three decellularization treatments maintained adequate mechanical and biochemical properties of B-ACL-B grafts to justify the further investigation of all three decellularization protocols. The selection of a superior treatment will depend on future studies of the propensity of treated tissues for repopulation by host ACL fibroblasts and, ultimately, on any immunogenic and/or remodeling host response induced in vivo.