Crosslinked Collagen Scaffolds Research Articles

Effect of radiation cross-linked collagen scaffold in alveolar ridge preservation of extraction socket.

This study aimed to evaluate the properties of radiation cross-linked collagen scaffold (RCS) and its efficacy for alveolar ridge preservation (ARP). RCS was prepared from collagen dispersion by electron beam irradiation and freeze-drying. The microstructure, swelling ratio, area alteration and mechanical properties of RCS were characterized. Fifty-four New Zealand rabbits performing incisor extraction on maxilla and mandible were randomly assigned into positive, sham operation or treatment groups. Micro-computed tomography (micro-CT) scans, performed after 1, 4, and 12 weeks of surgery, were to assess changes in ridge height at buccal and palatal side, in ridge width and in micromorphological parameters. Histological analysis accessed socket microarchitecture. The results showed that RCS had stable mechanical properties and morphologic features that provided a reliable physical support for ARP. Dimensional changes in treatment group revealed significantly greater vertical height at buccal (5.32 [3.37, 7.26] mm, p < .0001) and palatal (4.37 [2.66, 6.09] mm, p < .0001) side, and horizontal width at the maxilla (0.16 [0.04, 0.28] mm, p < .01) and mandible (0.33 [0.11, 0.54] mm, p < .01) than those in sham operation group after 12 weeks. The treatment group had advantage than positive group in vertical height preservation, quantitatively. The order and density of bone trabeculae were improved in treatment group. These findings indicated that RCS had the potential to serve as an effective scaffold for ARP.

Effects of various cross-linked collagen scaffolds on wound healing in rats model by deep-learning CNN

Scar tissue is connective tissue formed on the wound during the wound-healing process. The most significant distinction between scar tissue and normal tissue is the appearance of covalent cross-linking and the amount of collagen fibers in the tissue. This study investigates the efficacy of four types of collagen scaffolds in promoting wound healing and regeneration in a Sprague-Dawley murine model—the histomorphology analysis of collagen scaffolds and developing a deep learning model for accurate tissue classification. Four female rats (n = 24) groups received collagen scaffolds prepared through physical and chemical crosslinking. Wound healing progress was evaluated by monitoring granulation tissue formation, collagen matrix organization, and collagen fiber deposition, with histological scoring for quantification—the EDC and HA groups demonstrated enhanced tissue regeneration. The EDC and HA groups observed significant differences in wound regeneration outcomes. Deep-learning CNN models with data augmentation techniques were used for image analysis to enhance objectivity. The CNN architecture featured pre-trained VGG16 layers and global average pooling (GAP) layers. Feature visualization using Grad-CAM heatmaps provided insights into the neural network’s focus on specific wound features. The model’s AUC score of 0.982 attests to its precision. In summary, collagen scaffolds can promote wound healing in mice, and the deep learning image analysis method we proposed may be a new method for wound healing assessment.

Volumetric Outcomes of Peri-implant Soft Tissue Augmentation with a Xenogeneic Cross-Linked Collagen Scaffold: A Comparative Clinical Study.

Performing soft tissue augmentation (STA) at implant sites to improve esthetics, patient satisfaction, and peri-implant health is common. Several soft tissue grafting materials can be used to increase soft tissue thickness at the second-stage surgery, including human dermal matrices and xenogeneic collagen scaffolds. This study assessed and compared the volumetric outcomes, from second-stage surgery to crown delivery, around implants that received STA with a xenogeneic cross-linked collagen scaffold (XCCS) vs nonaugmented implant sites. Thirty-one patients (31 implant sites) completed the study. Intraoral digital scans were taken at the second stage and prior to crown delivery, and the STL files were imported in an image-analysis software to assess volumetric changes. XCCS-augmented implants showed significantly greater volumetric changes compared to control sites, which showed volume loss. The mean thickness of the XCCS-augmented area was 0.73 mm. There was no difference in patient-reported esthetic evaluations between groups. STA with XCCS provided significantly greater volumetric outcomes compared to nonaugmented sites. Further studies are needed to evaluate the long-term behavior of the augmented peri-implant mucosa and the effects of STA on peri-implant health.

Profitability of Chemically Cross-Linked Collagen Scaffold Production Using Bovine Pericardium: Revaluing Waste from the Meat Industry for Biomedical Applications

The meat industry generates a large amount of waste that can be used to create useful products such as bio-implants, which are usually expensive. In this report, we present an economic analysis of a continuous process for large-scale chemically cross-linked collagen scaffold (CCLCS) production in a Mexican context. For this purpose, three production capacities were simulated using SuperPro Designer® v 12.0: 5, 15, and 25 × 103 bovine pericardium units (BPU) per month as process feedstock. Data indicated that these capacities produced 2.5, 7.5, and 12.5 kg of biomesh per batch (per day), respectively. In addition, Net Unit Production Costs (NUPC) of 784.57, 458.94, and 388.26 $USD.kg−1 were obtained, correspondingly, with selling prices of 0.16 ± 0.078 USD.cm−2, 0.086 ± 0.043 USD.cm−2, and 0.069 ± 0.035 USD.cm−2, in the same order. We found that these selling prices were significantly lower than those in the current market in Mexico. Finally, distribution of costs associated with the process followed the order: raw materials &gt; facility-dependent &gt; labor &gt; royalties &gt; quality analysis/quality control (QA/QC) &gt; utilities. The present study showed the feasibility of producing low-cost and highly profitable CCLCS with a relatively small investment. As a result, the circular bioeconomy may be stimulated.

Preparation and Characterization of EDC/NHS Crosslinked Collagen Scaffold from the Scales of Cyprinus carpio (Common Carp)

Aquaculture is the fast-growing industry and, consequently, fish waste management is becoming a rising concern. Fish scale trash contains collagen as a biomaterial which offers excellent properties such as biocompatibility, biodegradability, porosity, low toxicity, cell adhesion and water absorption capacity which makes it suitable for biomedical research in the form of scaffolds. But the scaffolds have the limitation of having less stability. To overcome this shortcoming, collagen scaffolds can either be modified by chemical or physical treatments such as crosslinking with suitable polymers and dehydrothermal treatment, respectively. In the present study, extraction of fish collagen from the scales of Cyprinus carpio was performed followed by crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) & N-hydroxysuccinimide (NHS). Comparative characterization was done by techniques such as ATR-FTIR, FESEM and TGA to analyse the chemical composition, surface morphology and thermal degradation of fish collagen scaffold (CS) with EDC/NHS crosslinked collagen scaffold (EDC/NHS-CS). Crosslinked collagen scaffold’s antimicrobial activity was also determined with agar well diffusion assay by using S. aureus strain of bacteria and cytotoxicity on mouse fibroblast cells (NIH/3T3) by MTT assay. Moreover, the biomedical industry wants to shift to sustainable approaches and fish collagen-based scaffolds may prove to be a potential source in tissue engineering. And thus, also help in the management of fish waste.

Plasma-Polymerised Antibacterial Coating of Ovine Tendon Collagen Type I (OTC) Crosslinked with Genipin (GNP) and Dehydrothermal-Crosslinked (DHT) as a Cutaneous Substitute for Wound Healing

Tissue engineering products have grown in popularity as a therapeutic approach for chronic wounds and burns. However, some drawbacks include additional steps and a lack of antibacterial capacities, both of which need to be addressed to treat wounds effectively. This study aimed to develop an acellular, ready-to-use ovine tendon collagen type I (OTC-I) bioscaffold with an antibacterial coating for the immediate treatment of skin wounds and to prevent infection post-implantation. Two types of crosslinkers, 0.1% genipin (GNP) and dehydrothermal treatment (DHT), were explored to optimise the material strength and biodegradability compared with a non-crosslinked (OTC) control. Carvone plasma polymerisation (ppCar) was conducted to deposit an antibacterial protective coating. Various parameters were performed to investigate the physicochemical properties, mechanical properties, microstructures, biodegradability, thermal stability, surface wettability, antibacterial activity and biocompatibility of the scaffolds on human skin cells between the different crosslinkers, with and without plasma polymerisation. GNP is a better crosslinker than DHT because it demonstrated better physicochemical properties (27.33 ± 5.69% vs. 43 ± 7.64% shrinkage), mechanical properties (0.15 ± 0.15 MPa vs. 0.07 ± 0.08 MPa), swelling (2453 ± 419.2% vs. 1535 ± 392.9%), biodegradation (0.06 ± 0.06 mg/h vs. 0.15 ± 0.16 mg/h), microstructure and biocompatibility. Similarly, its ppCar counterpart, GNPppCar, presents promising results as a biomaterial with enhanced antibacterial properties. Plasma-polymerised carvone on a crosslinked collagen scaffold could also support human skin cell proliferation and viability while preventing infection. Thus, GNPppCar has potential for the rapid treatment of healing wounds.

Optimizing the combined soft tissue repair and osteogenesis using double surfaces of crosslinked collagen scaffolds.

Excessive tissue damage or loss has been solved by guided tissue regeneration and guided bone regeneration theories. However, the unfavorable degradation property of the resorbable collagen scaffold brings a big challenge to support soft tissue stabilization and time-consuming osteogenesis. The combined effect for soft tissue and bone of the collagen scaffold with better degradation pattern has not been clearly proven. This study determined whether the double surfaces of crosslinked collagen scaffolds could optimize the combined soft tissue repair and osteogenesis. In this study, we applied the chemically crosslinking treatment to the commercially available collagen scaffolds. Surface characterization, mechanical property and cell proliferation in vitro were evaluated. Combined bilateral skin and bone defects were established with the smooth surface of scaffold facing the skin defect and the rough surface facing the bone defect on the calvaria of rat. Micro-CT and histological evaluation were applied to determine the scaffold degradation pattern, soft tissue repair and osteogenesis. The crosslinked collagen scaffolds showed comparably favorable surface porosity, structure intactness, superhydrophilicity and mechanical properties. Compared to the native scaffolds, the crosslinked scaffolds could optimize the combined soft tissue repair and osteogenesis by preferably prolonged degradation time. Early pro-angiogenesis facilitated soft tissue repair and osteogenesis by upregulated soft tissue matrix degradation and balanced pro-osteogenesis with limited osteoclast-mediated bone resorption. Taken together, this study offers a promising repair strategy for the combined soft tissue and bone defects. Further, the possible mechanism of controllable scaffold degradation should be conducted.

Cross-Linked Collagen Scaffold from Fish Skin as an Ideal Biopolymer for Tissue Engineering

Cross-Linked Collagen Scaffold from Fish Skin as an Ideal Biopolymer for Tissue Engineering

Pneumatospinning Biomimetic Scaffolds for Meniscus Tissue Engineering.

Nanofibrous scaffolds fabricated via electrospinning have been proposed for meniscus tissue regeneration. However, the electrospinning process is slow, and can only generate scaffolds of limited thickness with densely packed fibers, which limits cell distribution within the scaffold. In this study, we explored whether pneumatospinning could produce thicker collagen type I fibrous scaffolds with higher porosity, that can support cell infiltration and neo-fibrocartilage tissue formation for meniscus tissue engineering. We pneumatospun scaffolds with solutions of collagen type I with thicknesses of approximately 1 mm in 2 h. Scanning electron microscopy revealed a mix of fiber sizes with diameters ranging from 1 to 30 µm. The collagen scaffold porosity was approximately 48% with pores ranging from 7.4 to 100.7 µm. The elastic modulus of glutaraldehyde crosslinked collagen scaffolds was approximately 45 MPa, when dry, which reduced after hydration to 0.1 MPa. Mesenchymal stem cells obtained from the infrapatellar fat pad were seeded in the scaffold with high viability (>70%). Scaffolds seeded with adipose-derived stem cells and cultured for 3 weeks exhibited a fibrocartilage meniscus-like phenotype (expressing COL1A1, COL2A1 and COMP). Ex vivo implantation in healthy bovine and arthritic human meniscal explants resulted in the development of fibrocartilage-like neotissues that integrated with the host tissue with deposition of glycosaminoglycans and collagens type I and II. Our proof-of-concept study indicates that pneumatospinning is a promising approach to produce thicker biomimetic scaffolds more efficiently that electrospinning, and with a porosity that supports cell growth and neo-tissue formation using a clinically relevant cell source.

Early soft tissue changes following implant placement with or without soft tissue augmentation using a xenogeneic cross‐link collagen scaffold: A volumetric comparative study

Soft tissue augmentation (STA) at implant sites has the potential of improving peri-implant health, esthetics, and marginal bone level stability. The present study aimed at evaluating the volumetric changes occurring following implant placement in sites that received STA compared to non-augmented sites. A total of 26 subjects received a dental implant in a posterior edentulous site. Simultaneous STA with a xenogeneic cross-linked collagen scaffold was performed for the first 13 patients, while the remaining subjects served as the negative control. An intraoral optical scanner was used at baseline and at 12 weeks to generate digital models. The mean volume (Vol) gain of the test group was 38.43 mm3 , while a mean Vol of -16.82 mm3 was observed for the control group (p <0.05). The mean thickness of the reconstructed volume (ΔD) was 0.61 and -0.24 mm, for the test and control group, respectively (p <0.05). Higher linear dimensional changes were observed for the test group (p <0.05), while no significant differences were observed in terms of keratinized mucosa width and pocket depth changes between the two groups. Simultaneous STA with xenogeneic collagen scaffold obtained statistically significant higher volumetric outcomes compared to the non-augmented group. STA at the time of implant placement using a xenogeneic cross-linked collagen scaffold can prevent remodeling of the ridge during the first 12 weeks, as compared to non-grafted implant sites.

Development of islet organoids from human induced pluripotent stem cells in a cross-linked collagen scaffold

Islets organoids would have value in the cell replacement therapy for diabetes apart from usual personalized drug screening routes. Generation of a large number of Islets like clusters, with ability to respond to glucose stimulation appears to be an ideal choice. In this study we have generated islet organoids with the ability to respond to glucose stimulation by insulin release. The source of the cells was an iPSC cell line differentiated into the pancreatic progenitors. These cells were assembled in matrigel or cross-linked collagen scaffold and compared for their efficacy to release insulin upon stimulation with glucose. The assembled organoids were examined by immunohistochemistry and expression of the relevant marker genes. The organoids showed expression of islet like markers in both - matrigel and crosslinked collagen scaffold. The islet organoids in both the cases showed release of insulin upon stimulation with glucose. The crosslinked collagen scaffold is quite stable and supports islet cells growth and function.

Dendrimer-Functionalized Metal Oxide Nanoparticle-Mediated Self-Assembled Collagen Scaffold for Skin Regenerative Application: Function of Metal in Metal Oxides.

Functionalized metal oxide nanoparticles cross-linked collagen scaffolds are widely used in skin regenerative applications because of their enhanced physicochemical and biocompatibility properties. From the safety clinical trials point of view, there are no reports that have compared the effects of functionalized metal oxide nanoparticles mediated collagen scaffolds for in vivo skin regenerative applications. In this work, triethoxysilane-poly (amido amine) dendrimer generation 3 (TES-PAMAM-G3 or G3)-functionalized spherical shape metal oxide nanoparticles (MO NPs: ZnO, TiO2, Fe3O4, CeO2, and SiO2, size: 12-25nm) cross-linked collagen scaffolds were prepared by using a self-assembly method. Triple helical conformation, pore size, mechanical strength, and in vitro cell viability of MO-TES-PAMAM-G3-collagen scaffolds were studied through different methods. The in vivo skin regenerative proficiency of MO-TES-PAMAM-G3-collagen scaffolds was analyzed by implanting the scaffold on wounds in Wistar albino rats. The results demonstrated that MO-TES-PAMAM-G3-collagen scaffold showed superior skin regeneration properties than other scaffolds. The skin regenerative efficiency of MO NPs followed the order ZnO > TiO2 > CeO2 > SiO2 > Fe3O4 NPs. This result can be attributed to higher mechanical strength, cell viability, and better antibacterial activity of ZnO-TES-PAMAM-G3-collagen scaffold that leads to accelerate the skin regenerative properties in comparison to other metal oxide based collagen scaffolds.

Mechanical characterization of rose bengal and green light crosslinked collagen scaffolds for regenerative medicine.

Collagen is one of the most important biomaterials for tissue engineering approaches. Despite its excellent biocompatibility, it shows the non-negligible disadvantage of poor mechanical stability. Photochemical crosslinking with rose bengal and green light (RGX) is an appropriate method to improve this property. The development of collagen laminates is helpful for further adjustment of the mechanical properties as well as the controlled release of incorporated substances. In this study, we investigate the impact of crosslinking and layering of two different collagen scaffolds on the swelling behavior and mechanical behavior in micro tensile tests to obtain information on its wearing comfort (stiffness, strength and ductility). The mechanical stability of the collagen material after degradation due to cell contact is examined using thickness measurements. There is no linear increase or decrease due to layering homologous laminates. Unexpectedly, a decrease in elongation at break, Young’s modulus and ultimate tensile strength are measured when the untreated monolayer is compared to the crosslinked one. Furthermore, we can detect a connection between stability and cell proliferation. The results show that with variation in number and type of layers, collagen scaffolds with tailored mechanical properties can be produced. Such a multi-layered structure enables the release of biomolecules into inner or outer layers for biomedical applications.

MP52-08 MACROPHAGE THERAPY VIA COLLAGEN BIOTEXTILE FOR THE TREATMENT OF STRESS URINARY INCONTINENCE

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Female Incontinence: Therapy II (MP52)1 Sep 2021MP52-08 MACROPHAGE THERAPY VIA COLLAGEN BIOTEXTILE FOR THE TREATMENT OF STRESS URINARY INCONTINENCE Ilaha Isali, Snigdha Cingireddi, Phillip McClellan, Mukesh Jain, James Anderson, Ozan Akkus, and Adonis Hijaz Ilaha IsaliIlaha Isali More articles by this author , Snigdha CingireddiSnigdha Cingireddi More articles by this author , Phillip McClellanPhillip McClellan More articles by this author , Mukesh JainMukesh Jain More articles by this author , James AndersonJames Anderson More articles by this author , Ozan AkkusOzan Akkus More articles by this author , and Adonis HijazAdonis Hijaz More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002082.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recent reviews1,2 proposed the use of macrophages (MΦ) for regenerative purposes either by way of modifying biomaterials to attract host macrophages, or by direct delivery of MΦ. We have demonstrated the polarization of M0 macrophages to the pro-regenerative subtype-M2 upon seeding on genipin crosslinked collagen scaffold (GES). Current study aimed assess the long-term tissue response and post-surgical mechanical properties of implanted GES seeded with M0, M1, and M2 in a rat model. METHODS: Monocytes were harvested from rat bone-marrow. M0, M1 or M2 macrophages were polarized from monocytes and MΦ subtypes were confirmed by flow cytometry. Electrochemically aligned collagen threads were filament wound to produce small collagen scaffold genipin crosslinked. M0, M1 or M2 macrophages were seeded on GES and implanted subcutaneously in rats to determine whether MΦ supplementation improves the stiffness (i.e modulus) of regenerated tissue, collagen deposition by host, and alters the biology of healing. Implants were recovered at 3 months following which they were tested in tension, cellularity was quantified in H&E-stained sections, collagen production was quantified by automated image analysis of Masson’s trichrome sections, and fibrous capsule thickness was measured. RESULTS: M2-subtype seeded scaffolds (GES+M2) had the greatest modulus such that its magnitude was about 2.5 times greater than that of cell-free scaffolds (GES) whereas modulus of GES+M0 or GES+M1 did not differ significantly from that of cell-free scaffolds. Number of cells counts per unit area was the greatest for GES+M2 group, suggesting a greater degree of host cell attraction. Furthermore, alpha smooth muscle actin, a marker of myofibroblasts, was more notably present in M2-seeded scaffolds. M2 delivery induced greater amount of collagen deposition while limiting fibrous encapsulation of the scaffold. Net effect of M2-delivery was such that improved healing biomechanics that converged closer to the 3-5 MPa native tissue modulus range that the scaffold alone is unable to converge. CONCLUSIONS: In vivo delivery of M2-subtype by filament wound collagen scaffolds results in superior healing biomechanics and implant remodelling in comparison to other macrophage subtypes. Source of Funding: This work is supported by NIH grant: R21HD095439-01

Optimization of Collagen Chemical Crosslinking to Restore Biocompatibility of Tissue-Engineered Scaffolds.

Collagen scaffolds, one of the most used biomaterials in corneal tissue engineering, are frequently crosslinked to improve mechanical properties, enzyme tolerance, and thermal stability. Crosslinkers such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) are compatible with tissues but provide low crosslinking density and reduced mechanical properties. Conversely, crosslinkers such as glutaraldehyde (GTA) can generate mechanically more robust scaffolds; however, they can also induce greater toxicity. Herein, we evaluated the effectivity of double-crosslinking with both EDC and GTA together with the capability of sodium metabisulfite (SM) and sodium borohydride (SB) to neutralize the toxicity and restore biocompatibility after crosslinking. The EDC-crosslinked collagen scaffolds were treated with different concentrations of GTA. To neutralize the free unreacted aldehyde groups, scaffolds were treated with SM or SB. The chemistry involved in these reactions together with the mechanical and functional properties of the collagen scaffolds was evaluated. The viability of the cells grown on the scaffolds was studied using different corneal cell types. The effect of each type of scaffold treatment on human monocyte differentiation was evaluated. One-way ANOVA was used for statistical analysis. The addition of GTA as a double-crosslinking agent significantly improved the mechanical properties and enzymatic stability of the EDC crosslinked collagen scaffold. GTA decreased cell biocompatibility but this effect was reversed by treatment with SB or SM. These agents did not affect the mechanical properties, enzymatic stability, or transparency of the double-crosslinked scaffold. Contact of monocytes with the different scaffolds did not trigger their differentiation into activated macrophages. Our results demonstrate that GTA improves the mechanical properties of EDC crosslinked scaffolds in a dose-dependent manner, and that subsequent treatment with SB or SM partially restores biocompatibility. This novel manufacturing approach would facilitate the translation of collagen-based artificial corneas to the clinical setting.

The Effect of Crosslinking Agents on the Properties of Type II Collagen Biomaterials

Type II collagen has been perceived as the indispensable element and plays a crucial role in cartilage tissue engineering. Thus, materials based on type II collagen have drawn farther attention in both academic and research for developing new systems for the cartilage regeneration. The disadvantage of using type II collagen as a biomaterial for tissue repairing is its reduced biomechanical properties. This can be solved by physical, enzymatic or chemical cross-linking processes, which provide biomaterials with the required mechanical properties for medical applications. To enhance type II collagen properties, crosslinked collagen scaffolds with different cross-linking agents were prepared by freeze-drying technique. The present research work studied the synthesis of type II collagen biomaterials with and without crosslinking agents. Scaffolds morphology was observed by MicroCT, showing in all cases an appropriate microstructure for biological applications, and the mechanical studies were performed using compressive tests. DSC showed an increase in denaturation temperature with an increase in cross-linking agent concentration. FTIR suggested that the secondary structure of collagen is not affected after the cross-linking; supplementary, to confirm the characteristic triple-helix conformation of collagen, the CD investigation was performed. The results showed that the physical-chemical properties of type II collagen were improved by cross-linking treatments.

Buccal tissue augmentation with a sugar cross‐linked collagen matrix: a case series study

Background: The thickness of buccal hard and soft tissue has been claimed to be of great clinical relevance in terms of aesthetics and health around implants. To improve such condition several surgical techniques have been proposed and several biomaterials have been used. Collagen products, cross-linked through the use of sugar according to GLYMATRIX technology, have shown their ability to undergo ossification in both animal and human studies, maintaining space for regeneration for long time. Aim/Hypothesis: To evaluate, through a series of consecutively treated patients, the ability of a GLYMATRIX collagen scaffold to improve buccal tissue thickness when used simultaneously to implant positioning in native bone. Materials and Methods: 16 patients, pre-operatively showing adequate bone for implant placement in a CBCT and flat\concave horizontal profile of the ridge in a clinical evaluation, were included in the study. Preoperative virtual models were developed using an intraoral scanner and a dedicated software. All patients received one or two implants and a cross-linked collagen scaffold (OSSIX Volumax, Datum Dental) was placed on the buccal bone plate at the same time. Flaps were repositioned to cover the matrix and sutured around a healing cap. After a conventional healing protocol (post-operative home care, suture removal, clinical and radiographic follow up visits) implants were loaded at 6 months and a new virtual model was obtained with the procedure of above. Gain in tissue thickness buccally to the implant was measured, by means of a computed superimposition of pre- and 6-month virtual models, as the largest improvement along the first 5 mm vertically measured from the gingival margin. Results: 20 implants with sugar cross-linked collagen matrix on the buccal side were consecutively placed in 16 patients. The mean buccal tissue gain at 6 months was 2.4 mm (SD = 0.7 mm). Conclusions and Clinical Implications: The use of a sugar cross-linked collagen matrix showed very promising results in improving buccal tissue thickness around implants. Such ability could be of high clinical relevance to improve this clinical parameter so important for health and esthetics with no needs for major procedures for augmentation. Keywords: Buccal tissue thickness, Tissue augmentation, Cross linked collagen matrix

Highly biocompatible novel polyphenol cross-linked collagen scaffold for potential tissue engineering applications

Collagen-based biomaterials are known to be the most potential and promising materials with diverse applications in tissue engineering and regeneration, due to its excellent biocompatibility. The major drawback of collagen is its rapid degradation, which can be overcome by crosslinking methods. Polyphenols exhibit excellent antioxidant activity and antimicrobial properties. In the present work, polyphenol-treated collagen scaffold (PPCS) was prepared by treating the aqueous extract of the dried ripe fruits of Terminalia chebula with bovine collagen. Scanning electron microscopy (SEM) of PPCS showed highly oriented interwoven fibrillar collagenous bundles in the network form. The chemical structure modification in PPCS was demonstrated by Fourier Transform Infrared spectroscopy. Tensile property and hydrophilicity of the PPCS showed enhanced values compared to the untreated collagen (COL). The thermal properties of COL and PPC scaffolds investigated by thermogravimetric analysis and differential scanning calorimetry, showed increased thermal decomposition and denaturation temperature values for the polyphenol crosslinked collagen. Further, the PPCS exhibited increased resistance to hydrolysis by collagenase. In vitro studies on biocompatibility showed increased fibroblast cell proliferation on PPCS. Thereby, the physico-chemical and biological properties of PPCS identify it as a promising scaffold with high biocompatibility for applications in tissue engineering and regenerative medicine.

Characterizing the Macrophage Response to Immunomodulatory Biomaterials Through Gene Set Analyses

The primary regulators of the innate immune response to implanted biomaterials are macrophages, which change phenotype over time to regulate multiple phases of the tissue repair process. Immunomodulatory biomaterials that target macrophage phenotype are a promising approach for promoting tissue repair. Although expression of multiple markers has been widely used to characterize macrophage phenotype, the complexity of the macrophage response to biomaterials makes interpretation difficult. The aim of this study was to put forth an objective method to characterize macrophage phenotype with respect to specific biological processes or standard phenotypes of interest. We investigated the utility of gene set analyses to analyze macrophages as they respond to model biomaterials in comparison to "reference" M1 and M2a macrophage phenotypes. Primary human macrophages were seeded onto crosslinked collagen scaffolds with or without adsorption of the proinflammatory cytokine interferon-gamma (IFNg). Gene expression of a custom-curated panel of 48 genes, representing the M1 and M2a gene signatures as well as other genes important for angiogenesis and tissue repair, was quantified using NanoString on days 3, 5, and 8 of culture. A dataset of phenotype controls, consisting of M0, M1, and M2a macrophages, was used as a source of comparison and to validate the methods of characterization. Gene expression of M1 and M2a markers showed mixed upregulation and downregulation by macrophages seeded on collagen and IFNg-adsorbed collagen scaffolds, highlighting the need for more holistic analyses. Euclidean distance measurements to the reference phenotypes were unable to resolve differences between groups. In contrast, rotation gene set testing with and without gene weighting based on the genes' ability to differentiate between M1, M2a, and M0 controls, followed by gene set variation analysis, showed that collagen scaffolds inhibited the classic M1 phenotype without promoting a classic M2a phenotype, and that IFNg-adsorbed collagen scaffolds promoted the M1 phenotype and inhibited the M2a phenotype. In summary, this work demonstrates a powerful, objective methodology for characterizing the macrophage response to biomaterials in comparison to reference macrophage phenotypes. With the addition of more macrophage phenotypes with defined gene expression signatures, this method could prove beneficial for characterizing complex hybrid phenotypes.

In Vitro Cultivation of Limbal Epithelial Stem Cells on Surface-Modified Crosslinked Collagen Scaffolds.

Purpose To investigate the efficacy of recombinant human collagen type I (RHC I) and collagen-like peptide (CLP) hydrogels as alternative carrier substrates for the cultivation of limbal epithelial stem cells (LESC) under xeno-free culture conditions. Methods Human LESC were cultivated on seven different collagen-derived hydrogels: (1) unmodified RHC I, (2) fibronectin-patterned RHC I, (3) carbodiimide-crosslinked CLP (CLP-12 EDC), (4) DMTMM- (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium-) crosslinked CLP (CLP-12), (5) fibronectin-patterned CLP-12, (6) “3D limbal niche-mimicking” CLP-12, and (7) DMTMM-crosslinked CLP made from higher CLP concentration solution. Cell proliferation, cell morphology, and expression of LESC markers were analyzed. All data were compared to cultures on human amniotic membrane (HAM). Results Human LESC were successfully cultivated on six out of seven hydrogel formulations, with primary cell cultures on CLP-12 EDC being deemed unsuccessful since the area of outgrowth did not meet quality standards (i.e., inconsistence in outgrowth and confluence) after 14 days of culture. Upon confluence, primary LESC showed high expression of the stem cell marker ΔNp63, proliferation marker cytokeratin (KRT) 14, adhesion markers integrin-β4 and E-cadherin, and LESC-specific extracellular matrix proteins laminin-α1, and collagen type IV. Cells showed low expression of differentiation markers KRT3 and desmoglein 3 (DSG3). Significantly higher gene expression of KRT3 was observed for cells cultured on CLP hydrogels compared to RHC I and HAM. Surface patterning of hydrogels influenced the pattern of proliferation but had no significant effect on the phenotype or genotype of cultures. Overall, the performance of RHC I and DMTMM-crosslinked CLP hydrogels was equivalent to that of HAM. Conclusion RHC I and DMTMM-crosslinked CLP hydrogels, irrespective of surface modification, support successful cultivation of primary human LESC using a xeno-free cultivation protocol. The regenerated epithelium maintained similar characteristics to HAM-based cultures.