In Vitro Characterization of Fat Grafts Processed Using the REVOLVE ENVI System versus Decantation.

Background: This preclinical study evaluated benchtop/in vitro properties and fat viability and activity of grafts processed using the REVOLVE ENVI 600 system compared with decantation and evaluated properties of REVOLVE ENVI waste. Methods: Lipoaspirate from six donors was processed using REVOLVE ENVI or decantation. The composition of each graft, hematocrit/red blood cell content, fat particle size/macrostructure, viable adipocyte count, and adipocyte activity were analyzed. Stromal vascular fraction was analyzed for viable progenitor cell count and colony-forming units. Results: REVOLVE ENVI grafts had a higher mean (±SD) fat content at 85.6% ± 6.1% than decanted grafts at 72.1% ± 4.0% (P < 0.001), with negligible free oil (0.4% ± 1.1%) and cellular debris (<0.1%), whereas REVOLVE ENVI waste contained primarily aqueous fluid (91.0% ± 2.2%) with negligible viable fat. REVOLVE ENVI grafts had significantly lower hematocrit levels (P < 0.001) and contained significantly more large fat globules (P < 0.001) than decanted grafts or REVOLVE ENVI waste. The percentage of tissue particles of more than 1000 µm was highest for REVOLVE ENVI grafts at 61.6% ± 9.2% (decantation: 52.5% ± 13.4%; REVOLVE ENVI waste: 0.49% ± 1.50%), and the percentage of particles less than 200 µm was lowest for REVOLVE ENVI grafts at 15.7% ± 2.6% (decantation: 32.2% ± 8.9%; REVOLVE ENVI waste: 97.9% ± 4.5%). REVOLVE ENVI grafts contained 145.2% ± 36.0% more viable adipocytes, 145.7% ± 46.2% greater activity, 195.5% ± 104.2% more progenitors in SVF, and 363.5% ± 161.2% more SVF colony-forming units than decanted grafts. Conclusion: Fat grafts processed using REVOLVE ENVI demonstrated greater viability and activity than decanted grafts in vitro.

Betadine Soaking of Silicone Coupons Minimally Impacts Acellular Dermal Matrix Incorporation in a Preclinical Primate Model.

BACKGROUND: Microbial pathogens local to prosthetic breast devices may promote infection, inflammation, and capsular contracture. Although antimicrobial solutions have been used, their effects on human acellular dermal matrix (HADM) incorporation when used with prosthetic devices are unknown. The authors' objective was to histologically assess the effect of 10% povidone iodine (PI)-saturated tissue expander (TE) exposure on HADM biological response in a primate model. They hypothesized that PI exposure would not negatively affect the HADM biological response. METHODS: Samples (1.5 × 1.5 cm) from smooth silicone TEs were saturated in saline or PI for 2 minutes and sutured to HADM to create HADM/TE constructs. Primates implanted subcutaneously with saline ( n = 9) and PI-treated HADM/TE ( n = 9) construct pairs were evaluated histologically for biological response after 2 or 4 weeks by means of a host response scoring scale (1 to 9), including recellularization, neovascularization, and inflammation. Inflammatory cells (eosinophils, lymphocytes, neutrophils, histiocytes, foreign-body giant cells) and evidence of HADM remodeling (fibroblasts, vessels) were further evaluated by means of a cell-specific scoring scale (0 to 4) and corroborated by immunostaining (CD3, CD20, CD68, FSP-1, collagen type IV). RESULTS: Mean histology scores were similar between saline- and PI-exposed HADM at 2 weeks (5.3 ± 0.9 and 5.6 ± 0.5; P = 0.52) and 4 weeks (4.6 ± 1.0 and 4.2 ± 0.9; P = 0.44). There was no difference in inflammatory cell presence at 2 and 4 weeks between groups. Fibroblast infiltration differences were insignificant between groups but exhibited trends toward an increase between time points for saline (1.6 ± 0.7 to 1.8 ± 0.8) and PI (1.3 ± 0.8 to 1.8 ± 1.0) groups, suggesting HADM incorporation over time. CONCLUSION: Data suggest that HADM exposure to PI-treated TEs does not negatively affect inflammation, vascularization, recellularization, incorporation, or host response to HADM in this model. CLINICAL RELEVANCE STATEMENT: PI is a surgical pocket irrigant used to address bacterial colonization, but its impact on ADM incorporation is unknown. This study demonstrates similar biologic response to ADMs adjacent to PI- or saline-saturated TEs in a primate model.

Acellular Dermal Matrix Susceptibility to Collagen Digestion: Effect on Mechanics and Host Response.

Various tissue origins and manufacturing processes can differentially affect the retention of native properties of acellular dermal matrices (ADMs); however, comparative studies are limited. Head-to-head comparisons between different configurations of porcine-derived Strattice (Allergan Aesthetics, an AbbVie Company, Irvine, CA) and bovine-derived SurgiMend (Integra LifeSciences, Billerica, MA) ADMs were performed to evaluate mechanical integrity and host tissue biologic response. Thermodynamic profile and morphology, which affect retention of mechanical strength, were evaluated through differential scanning calorimetry, scanning electron microscopy, and histology. Mechanical strength was assessed through tensile testing following collagenase exposure in vitro and following subcutaneous implantation in a rodent model. Host biologic response was evaluated through histopathology. Compared with respective native tissues, reductions in onset melting temperature following tissue processing were smaller for Strattice Firm versus SurgiMend 1.0 (Δ0.79°C vs. Δ5.77°C), Strattice Extra Thick versus SurgiMend 3.0 (Δ1.57°C vs. Δ4.79°C), and Strattice Perforated versus SurgiMend Microperforated (Δ1.18°C vs. Δ7.76°C), with similar trends for peak melting temperature. Strattice maintained native dermal architecture versus compacted collagen with process-induced interstices observed for SurgiMend. Strattice Firm, Extra Thick, and Perforated retained 33.44%, 65.65%, and 17.20% of initial strength after 48 h exposure to excess collagenase, while the SurgiMend ADMs were completely digested by 48 h. At 6 weeks postimplantation, both Strattice and SurgiMend showed minimal inflammatory response, but greater fibroblast repopulation was evident for Strattice. Strattice Firm had higher maximum load (145.85 ± 33.05 N/cm vs. 24.29 ± 12.35 N/cm, p ≤ 0.01), maximum stress (8.20 ± 1.91 MPa vs. 2.24 ± 1.27 Mpa, p ≤ 0.01), and stiffness (7491.00 ± 1981.32 N/cm vs. 737.56 ± 292.55 N/cm, p ≤ 0.01) than SurgiMend 1.0. Strattice Extra Thick had lower maximum load (198.54 ± 58.79 N/cm vs. 303.08 ± 76.76 N/cm, p < 0.05) than SurgiMend 3.0, but similar maximum stress (6.96 ± 1.78 Mpa vs. 8.73 ± 2.15 Mpa) and stiffness (13386.11 ± 3123.28 N/cm vs. 9389.02 ± 4860.67 N/cm). Strattice Perforated had higher maximum load (72.65 ± 41.44 N/cm vs. 10.23 ± 4.67 N/cm, p < 0.05) and maximum stress (4.08 ± 2.08 Mpa vs. 0.44 ± 0.19 p < 0.05) than SurgiMend Microperforated. Maximum load retention rates following implantation were higher for Strattice Firm versus SurgiMend 1.0 (37.85% vs. 8.03%), Strattice Extra Thick versus SurgiMend 3.0 (45.03% vs. 37.80%), and Strattice Perforated versus SurgiMend Microperforated (28.04% vs. 6.21%). Similar results were obtained for maximum stress and stiffness. In conclusion, Strattice retained greater mechanical strength in vitro and in vivo, while exhibiting greater fibroblast cell infiltration. Impact statement Acellular dermal matrix (ADM)-derived surgical meshes are used in abdominal wall reconstruction procedures, such as hernia repair. Comparative studies evaluating the mechanical properties of ADMs and how they integrate with host tissue are essential because these properties impact performance in a clinical setting. This study compared the maintenance of mechanical integrity and host tissue biologic response of two commercially available ADMs, Strattice and SurgiMend, using in vitro and in vivo techniques. A better understanding of the properties of ADMs is expected to impact mesh selection and help to minimize the incidence of herniation recurrence and need for revisional surgery.

Porcine bone-patellar tendon-bone xenograft in a caprine model of anterior cruciate ligament repair.

The use of human tissue-derived autografts and allografts continues to be the gold standard in anterior cruciate ligament (ACL) repair. However, autografts and allografts have their own set of associated risks. Many alternative options, including synthetic replacements, have failed to demonstrate long-term success. In this study, sterile acellular porcine bone-tendon-bone (BTB) xenografts were created using a proprietary process and tested against BTB autografts in goats for 13 and 52 weeks. At 13 weeks, all xenograft-implanted animals (n = 9) had subjective hind leg motor function (HLMF) that was categorized as either normal (score = 0) or a slight limp (score = 1) compared with two of nine autograft-implanted animals having a moderate limp (score = 2). At 39 weeks, there was HLMF improvement with each autograft-implanted and xenograft-implanted animal having normal HLMF or only a slight limp. At 13 weeks, six of nine animals in each group achieved normal anterior drawer scores, which increased to nine of nine animals in each group by 39 weeks. Both autografts and xenografts exhibited minimal inflammation with excellent integration of the fibrous tendon portion of the graft to host bone, as evidenced histologically by Sharpey's fiber formation. At 52 weeks, maximum mechanical load at failure for xenografts was 1092.0 ± 586.4 N compared with 1037.0 ± 422.6 N for autografts. These results demonstrate that a sterile acellular porcine BTB xenograft can perform equivalently to BTB autograft in a caprine model of ACL repair.

Relevant In Vitro Predictors of Human Acellular Dermal Matrix-Associated Inflammation and Capsule Formation in a Nonhuman Primate Subcutaneous Tissue Expander Model.

Objective: Benchtop methods were evaluated for preclinical inflammation/capsule formation correlation following implantation of human acellular dermal matrices. Methods: Dermal matrices were compared with native dermis for structure (histology, scanning electron microscopy), collagen solubility (hydroxyproline), enzymatic susceptibility (collagenase), and thermal stability (differential scanning calorimetry). Results were compared with implantation outcomes in a primate tissue expander model. Results: Native dermis, electron beam-sterilized, and freeze-dried human acellular dermal matrices had equivalent morphology, acid-soluble collagen (60.5% ± 6.3%, 65.3% ± 3.2%, and 63.3% ± 2.4%, respectively), and collagenase resistance. Implant results showed minimal inflammation/matrix degradation, lack of capsule formation, insignificant elastic modulus change (57.65 ± 20.24 MPa out-of-package/44.84 ± 23.87 MPa in vivo), and low antibody induction (2- to 8-fold increase) for electron beam-sterilized matrix. Similar results for freeze-dried dermal matrix were previously observed. γ-Irradiated, γ-irradiated/freeze-dried, and ethanol-stored dermal matrices were statistically different from native dermis for acid-soluble collagen (82.4% ± 5.8%, 72.2% ± 6.2%, and 76.8% ± 5.0%, respectively) and collagenase digestion rate, indicating matrix damage. γ-Irradiated matrix-implanted animals demonstrated elevated inflammatory response, foreign body giant cells, capsule formation at the tissue expander junction, and robust matrix metalloproteinase-1 staining with significant elastic modulus decrease (37.43 ± 7.52 MPa out-of-package/19.58 ± 1.16 MPa in vivo). Antibody increase (32- to 128-fold) was observed 6 to 10 weeks following γ-irradiated matrix implantation. Ethanol-stored dermal matrix elicited an acute antibody response (4- to 128-fold increase, 2-4 weeks) and macrophage-concentrated synovial-like hyperplasia at the tissue expander junction, moderate matrix metalloproteinase-1 staining, and significant elastic modulus decrease (61.15 ± 9.12 MPa out-of-package/17.92 ± 4.02 MPa in vivo) by 10 weeks implantation. Conclusion: Demonstrated loss of collagen integrity in vitro may be predictive of inflammation/capsule formation in primate tissue expander models. These results may be further predictive of clinical observations.

Comparative host response of 2 human acellular dermal matrices in a primate implant model.

OBJECTIVE: We examined the differences in capsule formation between 2 commercially available human acellular dermal matrices in a nonhuman primate model. METHODS: Primates were implanted dorsally with a subcutaneously placed tissue expander and randomized into 3 groups, receiving skin coverage only, coverage with non-irradiated freeze-dried human acellular dermal matrix, or coverage with gamma-irradiated human acellular dermal matrix. After 9 weeks, soft tissue around the tissue expander was excised and evaluated qualitatively and quantitatively to assess extent of inflammation (CD68 antibodies and interleukin-6 levels), degradation and fibrosis (matrix metalloproteinase-1 and procollagen-1 staining), and mechanical (tensile) strength. RESULTS: Histological evaluation of tissue around the tissue expander indicated differences in host response, suggesting capsule presence in the gamma-irradiated matrix group but not the freeze-dried matrix group. The extent of local inflammation was much higher in the gamma-irradiated matrix group which demonstrated mean (standard deviation) localized interleukin-6 concentration of 67.3 (53.6) vs 16.3 (6.7) pg/mg protein in the non-irradiated matrix group. There was robust degradation and fibrotic response in the gamma-irradiated matrix group versus the freeze-dried matrix group. Mechanical testing indicated mean (standard deviation) ultimate tensile strength of 12.0 (7.1) N in the gamma-irradiated matrix group versus 99.3 (48.8) N in the freeze-dried matrix group. CONCLUSIONS: Enclosure of a tissue expander with human acellular dermal matrix untreated by gamma irradiation led to minimal inflammation and minimal evidence of fibrosis/capsule around the tissue expander compared with robust capsule formation around the tissue expander that was covered by a gamma-irradiated human acellular dermal matrix.

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.

Implantation of a porcine acellular dermal graft in a primate model of rotator cuff repair.

BACKGROUND: Non-cross-linked xenogeneic extracellular matrix graft materials have typically elicited a hypersensitivity reaction when implanted into humans or other primates. The purpose of this study was to examine the histologic and immune response to a non-cross-linked porcine-derived dermal extracellular matrix graft processed to remove the α-gal epitope. MATERIALS AND METHODS: Eight African green monkeys were implanted with porcine acellular dermal matrix (Conexa Reconstructive Tissue Matrix; Tornier Inc, Edina, MN, USA) to repair and augment a partial excision defect of the supraspinatus tendon of the rotator cuff. Four animals each were sacrificed at 3 months and 6 months, and histologic samples were compared with tissues harvested from unoperated shoulders. RESULTS: Gross examination of grafted Conexa showed the appearance of integration proximally with tendon and distally with bone in each operated rotator cuff complex. Histologically, Conexa appeared to have remodeled to tendon-like architecture, with homogeneous distribution of fibroblast cells and parallel alignment of collagen fibers, with the direction of force evident by 3 months after implantation. Abundant vasculature observed at 3 months, which diminished to native tendon levels by 6 months, also indicated this to be a period of significant remodeling with an absence of significant inflammation, as evidenced by immunochemical methods and serum analysis. CONCLUSION: Conexa porcine acellular dermal matrix allows for incorporation of host tendon tissue without a hypersensitivity reaction in a primate model and should be a safe material for augmentation of human rotator cuff repair.

Retention of structural and biochemical integrity in a biological mesh supports tissue remodeling in a primate abdominal wall model.

AIM: Suboptimal clinical outcome following the implantation of porcine-derived tissue matrices may be due to the method of processing the material to achieve an acellular graft and to reduce the immune response to xenogeneic epitopes. The ability to produce a porcine-based graft material that retains the structural integrity of the extracellular matrix and minimizes the potential antigenic response to the galactose-alpha(1,3)-galactose terminal disaccharide (alpha-Gal) may allow the scaffold to support regeneration of native tissue. MATERIALS & METHODS: Porcine dermal tissue was processed to remove cells and DNA, and minimize the presence of alpha-Gal via specific enzymatic cleavage. In vivo performance was evaluated by implantation into the abdominal wall of an Old World primate exisional repair model. Grafts were explanted at 0.5, 1, 3 and 6 months and assessed for cellular repopulation and vascularization, for localized immune response by presence of T cells, B cells and macrophages, and systemic immune response by anti-alpha-Gal IgG by ELISA. RESULTS: Animals tolerated implants well and exhibited no clinical signs of inflammation, laxity, hernia or visceral tissue attachment. Histological evaluation revealed marked host fibroblast repopulation and neoangiogenesis as early as 2 weeks postimplant. Cellular repopulation and maturation of vascular structures reached a plateau at 3 months. Immunological evaluation of immune cell infiltration demonstrated an early, mixed cellular inflammatory response at 2 weeks. This cellular immune response was transient and diminished to baseline levels by 3 months postimplant. CONCLUSION: The combination of a nondamaging process, successful removal of cells, and reduction of the xenogeneic alpha-Gal antigens from the porcine dermal matrix, while maintaining an intact extracellular matrix, is critical to its ability to remodel and integrate into host tissue, leading to its overall acceptance.

Host response to human acellular dermal matrix transplantation in a primate model of abdominal wall repair.

Commercially available human acellular dermal matrix (HADM), AlloDerm((R)), was implanted as an interpositional graft in the abdominal wall of adult vervet monkeys. Host response to implanted HADM was evaluated and compared with a human cellular dermal matrix (HCDM) and a primate acellular dermal matrix (PADM). Clinical acceptance of the acellular grafts (HADM and PADM) and graft remodeling were evidenced by fibroblast repopulation and neoangiogenesis. A mild inflammatory response marked predominantly by macrophages and T-cells was present in both HADM and PADM during the first month but was absent by 3 months. Similarly, antibody and complement deposition into the grafts as well as in the serum was evident only at the early time points. Interleukin-6 (IL-6) or IL-10 was induced in some acellular graft-implanted monkeys at the early time points, but tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), or IL-2 was not detected over the study period. In contrast, significant inflammation was observed in HCDM-implanted animals, as evidenced by immune cell infiltration (p

Host response to implanted porcine-derived biologic materials in a primate model of abdominal wall repair.

Three commercially available porcine-derived biologic meshes were implanted in an Old World primate abdominal wall resection repair model to compare biological outcome as a predictor of clinical efficacy. Tissues were explanted over a 6-month period and evaluated for gross pathology, wound healing strength, mesenchymal cellular repopulation, vascularity, and immune response. In vivo functional outcomes were correlated with in vitro profile for each material. Small intestinal submucosa-based implants demonstrated scar tissue formation and contraction, coincident with mesh pleating, and were characterized by immediate and significant cellular and humoral inflammatory responses. Porcine dermal-based grafts demonstrated significant graft pleating, minimal integration, and an absence of cellular repopulation and vascularization. However, a significant cellular immune response surrounded the grafts, coincident with poor initial wound healing strengths. In vivo observations for the three porcine-derived mesh products correlated with individual in vitro profiles, indicating an absence of characteristic biochemical markers and structural integrity. This correlation suggests that in vivo results observed for these mesh products are a direct consequence of specific manufacturing processes that yield modified collagen matrices. The resulting loss of biological and structural integrity elicits a foreign body response while hindering normal healing and tissue integration.

A novel polyethylene depot device for the study of PLGA and P(FASA) microspheres in vitro and in vivo.

Polymer microspheres (0.5-5.0 microm) are difficult to characterize in vivo because they degrade, migrate, and are endocytosed. A novel polyethylene mesh pouch containing microspheres allowed for retrieval of degraded polymeric products from rats without affecting the rate of degradation. Pouches containing poly(lactic-co-glycolic acid) (PLGA) or poly(fumaric-co-sebacic acid) (P(FASA)) microspheres were implanted intramuscularly, subcutaneously, and intraperitoneally and analyzed after 3, 7, 14, and 28 days. In vivo, subcutaneous or intraperitoneal implants experienced an immediate mass loss and a delayed decrease in molecular weight (Mw). Intramuscular implants behaved similarly to in vitro samples, decreasing in Mw immediately and lagging in mass loss. These results suggest that mass loss, which is usually dependent on Mw loss in vitro, may be directly due to enzymatic, rather than hydrolytic, degradation subcutaneously and intraperitoneally, while intramuscular implants appear to be mostly dependent on hydrolytic cleavage. This observation is further supported by histology. Additional experiments on pouches loaded with PLGA microspheres encapsulating osteoprotegerin, a protein drug used to prevent bone resorption, revealed that use of the device prevented the artifactual polymer compression inherent to microsphere centrifugation during release studies and allowed for the extraction of active protein from microspheres implanted for 3 days in vivo.

Effect of lecithin and MgCO3 as additives on the enzymatic activity of carbonic anhydrase encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres.

A model enzyme, carbonic anhydrase, was encapsulated and released from poly(lactide-co-glycolide) (PLGA) microspheres (1-3 microm) made by a novel phase inversion technique. Lecithin was used as a surfactant in the encapsulation process and was incorporated in either the organic phase, aqueous phase, both phases, or not at all. Additional microspheres were also made with lecithin incorporated in the aqueous phase and a basic salt, MgCO3, in the polymeric phase. Released carbonic anhydrase, protein extracted from microspheres, or enzyme incubated with lecithin and PLGA were analyzed via HPLC and activity assay to determine the effect of these additives on protein integrity and activity. Lecithin in the aqueous phase appeared to increase the fraction of enzyme in monomeric form as well as its activity for both extracted protein and released protein as compared to the other formulations without MgCO3. Incubation of enzyme with PLGA degradation products indicated that the acidic environment within the microspheres aids in the irreversible inactivation of the encapsulated protein. Addition of MgCO3 further increased the amount of monomer in both the extracted and released protein by decreasing the amount of acid-induced cleavage and noncovalent aggregation, but still greatly decreased the activity of the enzyme.