We build long-term relationships by supporting and working closely with customers. If you have a question for our team of collagen scientists, please reach us at info@collagensolutions.com

collagen function resource library  Collagen Research Products and Services

Medical Device Applications for Collagen

Listing of collagen-based medical devices
Application: General Collagen

This brochure provides a overview of the numerous collagen-based medical device applications that Collagen Solutions can support.

Medical Grade Soluble Collagen Benefits

Advantages of working with GMP soluble bovine collagen
Application: Cell Delivery, Application: Drug Delivery, Application: General Collagen

This brochure summarizes the numerous soluble collagen applications and the benefits of working with a medical grade, highly purified collagen source. To order, click here.

Off-the-Shelf Collagen Products

Standard GMP collagen products for use in research
Application: Cell Delivery, Application: Drug Delivery, Application: General Collagen

This brochure describes all of the standard collagen products that are supplied by Collagen Solutions. To order, click here.

Regenerative Orthopaedic Solutions

Overview of capabilities for orthopaedic medical devices
Application: General Collagen

This brochure highlights our experience and expertise in the development of orthobiologics, including bovine bone chips, Bone Graft Substitute, collagen scaffolds, decellularized ECM, collagen coatings, collagen particles, dressings & grafts, and flowable matrices.

Safe & Reliable Bovine Pericardium for Medical Devices

Tissue sourced from bovine with the lowest possible risk of BSE
Application: Pericardium

This brochure highlights our Quality and Logisitics standards and capabilities of our tissue procurement, processing, and delivery. 

Collagen Function Technical Resources  Collagen Function Technical Resources

3D Gel Formation Protocol

Procedure to form a 3D collagen gel
Application: Cell Delivery, Application: Drug Delivery, Application: General Collagen

This video demonstrates the steps taken to form a 3D gel using our soluble collagen and titration buffer. To order, click here.

Atelo Soluble Collagen White Paper

Soluble collagen instructions for use
Application: Cell Delivery, Application: Drug Delivery, Application: General Collagen

This white paper provides atelo collagen specifications, storage requirements, and protocols for use. To order, click here.

Biomaterials to Progress Research to Clinic

Comparison of medical vs. research grade collagen
Application: Cell Delivery, Application: Drug Delivery, Application: General Collagen

This technical highlight summarizes the unique physical properties of collagen and the benefits of using medical grade collagen transition technologies into the clinic. To order, click here.

Bovine Tendon Long Fibre Powder White Paper

Lyophilized, long fibre polymeric collagen application overview
Application: Bone, Application: Bone/Cartilage, Application: Dental, Application: General Collagen

This white paper provides collagen specifications, storage requirements, and application details for long fibre milled powder from tendon.

Collagen for Cell Delivery

Literature review of collagen carriers for cells
Application: Cell Delivery, Application: General Collagen

This review provides an overview of numerous applications where collagen has been investigated as delivery vehicle for cell therapies.

Collagen for Drug Delivery

Literature review of collagen carriers for biomolecules
Application: Drug Delivery, Application: General Collagen

This review provides an overview collagen carriers for a variety of drug delivery applications.

Exclusive IP position for a Unique Pericardium Biomaterial

Unique Pericardium Biomaterial with Advantages for TAVR, TMVR & Other Transcatheter Cardiovascular Therapies
Application: Pericardium

This technical highlight summarizes the unique pericardium biomaterial properties and their advantages to percutaneous delivery devices for TAVR, TMVR & other transcatheter cardiovascular therapies. 

Fibrillar Collagen White Paper

Fibrillar collagen instructions for use
Application: Bone, Application: Cell Delivery, Application: Drug Delivery, Application: General Collagen, Application: Wound

This white paper provides fibrillar collagen specifications, storage requirements, and protocols for use.

Lyophilized Polymeric Collagen White Paper

Lyophilized polymeric collagen application overview
Application: Bone, Application: Bone/Cartilage, Application: Cartilage, Application: Dental, Application: General Collagen, Application: Tendon, Application: Tendon/Bone (Rotator Cuff), Application: Wound

This white paper provides collagen specifications, storage requirements, and application details for neutral polymeric powder from tendon.

Support for Bovine Collagen in Medical Devices

Justification for the use of bovine animal tissues in medical devices
Application: General Collagen

This literature review addresses the advantages of using collagen in medical devices and clarifies the perceived risks associated with the use of bovine sources.

Collagen Resources Publications  Collagen Resources Publications

A Gel Aspiration-Ejection System for the Controlled Production and Delivery of Injectable Dense Collagen Scaffolds

Kamranpour NO et al. 2016. Biofabrication. doi:10.1088/1758-5090/8/1/015018
Application: Bone

A gel aspiration-ejection (GAE) system has been developed for the advanced production and delivery of injectable dense collagen (I-DC) gels of unique collagen fibrillar densities (CFDs). Through the creation of negative pressure, GAE aspirates prefabricated highly hydrated collagen gels into a needle, simultaneously inducing compaction and meso-scale anisotropy (i.e., fibrillar alignment) on the gels, and by subsequent reversal of the pressure, I-DC gels can be controllably ejected. The system generates I-DC gels with CFDs ranging from 5 to 32 wt%, controlling the initial scaffold microstructure, anisotropy, hydraulic permeability, and mechanical properties. These features could potentially enable the minimally invasive delivery of more stable hydrogels. The viability, metabolic activity, and differentiation of seeded mesenchymal stem cells (MSCs) was investigated in the I-DC gels of distinct CFDs and extents of anisotropy produced through two different gauge needles. MSC osteoblastic differentiation was found to be relatively accelerated in I-DC gels that combined physiologically relevant CFDs and increased fibrillar alignment. The ability to not only support homogenous cell seeding, but also to direct and accelerate their differentiation through tissue-equivalent anisotropy, creates numerous opportunities in regenerative medicine.

A Methylcellulose and Collagen Based Temperature Responsive Hydrogel Promotes Encapsulated Stem Cell Viability and Proliferation In Vitro

Payne C et al. 2017. Drug Deliv & Transl Res. 7:132-146
Application: Cell Delivery

With the number of stem cell-based therapies emerging on the increase, the need for novel and efficient delivery technologies to enable therapies to remain in damaged tissue and exert their therapeutic benefit for extended periods, has become a key requirement for their translation. Hydrogels, and in particular, thermoresponsive hydrogels, have the potential to act as such delivery systems. Thermoresponsive hydrogels, which are polymer solutions that transform into a gel upon a temperature increase, have a number of applications in the biomedical field due to their tendency to maintain a liquid state at room temperature, thereby enabling minimally invasive administration and a subsequent ability to form a robust gel upon heating to physiological temperature. However, various hurdles must be overcome to increase the clinical translation of hydrogels as a stem cell delivery system, with barriers including their low tensile strength and their inadequate support of cell viability and attachment. In order to address these issues, a methylcellulose based hydrogel was formulated in combination with collagen and beta glycerophosphate, and key development issues such as injectability and sterilization processes were examined. The polymer solution underwent thermogelation at ~36 °C as determined by rheological analysis, and when gelled, was sufficiently robust to resist significant disintegration in the presence of phosphate buffered saline (PBS) while concomitantly allowing for diffusion of methylene blue dye solution into the gel. We demonstrate that human mesenchymal stem cells (hMSCs) encapsulated within the gel remained viable and showed raised levels of dsDNA at increasing time points, an indication of cell proliferation. Mechanical testing showed the “injectability”, i.e. force required for delivery of the polymer solution through devices such as a syringe, needle or catheter. Sterilization of the freeze-dried polymer wafer via gamma irradiation showed no adverse effects on the formed hydrogel characteristics. Taken together, these results indicate the potential of this gel as a clinically translatable delivery system for stem cells and therapeutic molecules in vivo.

A Micro-Architecturally Biomimetic Collagen Template for Mesenchymal Condensation Based Cartilage Regeneration

Younesi M et al. 2016. Acta Bio. 30: 212-221
Application: Bone/Cartilage, Application: Cartilage

The unique arcade-like orientation of collagen fibers enables cartilage to bear mechanical loads. In this study continuous-length aligned collagen threads were woven to emulate the interdigitated arcade structure of the cartilage. The weaving pattern provided a macropore network within which micromass cell pellets were seeded to take advantage of mesenchymal condensation driven chondrogenesis. Compression tests showed that the baseline scaffold had a modulus of 0.83 ± 0.39 MPa at a porosity of 80%. The modulus of pellet seeded scaffolds increased by 60% to 1.33 ± 0.37 MPa after 28 days of culture, converging to the modulus of the native cartilage. The scaffolds displayed duress under displacement controlled low-cycle fatigue at 15% strain amplitude such that load reduction stabilized at 8% after 4500 cycles of loading. The woven structure demonstrated a substantial elastic recoil where 40% mechanical strain was close to completely recovered following unloading. A robust chondrogenesis was observed as evidenced by positive staining for GAGs and type II collagen and aggrecan. Dimethyl methylene blue and sircol assays showed GAGs and collagen productions to increase from 3.36 ± 1.24 and 31.46 ± 3.22 at day 3 to 56.61 ± 12.12 and 136.70 ± 12.29 μg/μg of DNA at day 28 of culture. This woven collagen scaffold holds a significant potential for cartilage regeneration with shorter in vitro culture periods due to functionally sufficient mechanical robustness at the baseline. In conclusion, the mimicry of cartilage’s arcade architecture resulted in substantial improvement of mechanical function while enabling one of the first pellet delivery platforms enabled by a macroporous network.

Acellular Hydroxyapatite-Collagen Scaffolds Support Angiogenesis and Osteogenic Gene Expression in an Ectopic Murine Model: Effects of Hydroxyapatite Volume Fraction

Jeagher MJ et al. 2016. J Biomed Mater Res Part A. 104A: 2178-2188
Application: Bone

Acellular hydroxyapatite (HA) reinforced collagen scaffolds were previously reported to induce angiogenesis and osteogenesis after ectopic implantation but the effect of the HA volume fraction was not investigated. Therefore, the objective of this study was to investigate the effect of HA volume fraction on in vivo angiogenesis and osteogenesis in acellular collagen scaffolds containing 0, 20, and 40 vol % HA after subcutaneous ectopic implantation for up to 12 weeks in mice. Endogenous cell populations were able to completely and uniformly infiltrate the entire scaffold within 6 weeks independent of the HA content, but the cell density was increased in scaffolds containing HA versus collagen alone. Angiogenesis, remodeling of the original scaffold matrix, mineralization, and osteogenic gene expression were evident in scaffolds containing HA, but were not observed in collagen scaffolds. Moreover, HA promoted a dose-dependent increase in measured vascular density, cell density, matrix deposition, and mineralization. Therefore, the results of this study suggest that HA promoted the recruitment and differentiation of endogenous cell populations to support angiogenic and osteogenic activity in collagen scaffolds after subcutaneous ectopic implantation.

Age Dependent Differences in Collagen Alignment of Glutaraldehyde Fixed Bovine Pericardium

Sizeland KH et al. 2014. BioMed Res Int. 1-14
Application: Pericardium

Bovine pericardium is used for heart valve leaflet replacement where the strength and thinness are critical properties. Pericardium from neonatal animals (4–7 days old) is advantageously thinner and is considered as an alternative to that from adult animals. Here, the structures of adult and neonatal bovine pericardium tissues fixed with glutaraldehyde are characterized by synchrotron-based small angle X-ray scattering (SAXS) and compared with the mechanical properties of these materials. Significant differences are observed between adult and neonatal tissue. The glutaraldehyde fixed neonatal tissue has a higher modulus of elasticity (83.7 MPa) than adult pericardium (33.5 MPa) and a higher normalized ultimate tensile strength (32.9 MPa) than adult pericardium (19.1 MPa). Measured edge on to the tissue, the collagen in neonatal pericardium is significantly more aligned (orientation index (OI) 0.78) than that in adult pericardium (OI 0.62). There is no difference in the fibril diameter between neonatal and adult pericardium. It is shown that high alignment in the plane of the tissue provides the mechanism for the increased strength of the neonatal material. The superior strength of neonatal compared with adult tissue supports the use of neonatal bovine pericardium in heterografts. 

Biologic and Synthetic Grafts in the Reconstruction of Large to Massive Rotator Cuff Tears

Gillespie RJ et al. 2016. AAOS. 24(12): 823-828
Application: Tendon/Bone (Rotator Cuff)

Rotator cuff injuries are common in both young and elderly patients. Despite improvements in instrumentation and surgical techniques, the failure rates following tendon reconstruction remain unacceptably high. To improve outcomes, graft patches have been developed to provide mechanical strength and to furnish a scaffold for biologic growth across the delicate tendon-bone junction. Although no patch effectively re-creates the structured, highly organized system of prenatal tendon development, augmenting rotator cuff repair may help restore native tendon-to-bone attachment while reproducing the mechanical and biologic properties of native tendon. An understanding of biologically and synthetically derived grafts, along with knowledge of the preliminary data available regarding their combined use with growth factors and stem cells, is needed to improve management and treatment outcomes. The current literature has not been consistent in showing patch augmentation to be beneficial over traditional repair, but novel scaffolding materials may help facilitate rotator cuff tendon repair that is histologically and biomechanically comparable to native tendon.

Biomaterials with Enhanced Properties and Devices Made Therefrom

Cunanan et al. 2015 Patent No.: US 9095430B2
Application: Pericardium

Biomaterials with enhanced properties such as improved strength, flexibility, durability and reduced thickness are use ful in the fabrication of biomedical devices, particularly those Subjected to continuous or non-continuous loads where repeated flexibility and long-term durability are required. These enhanced properties can be attributed to elevated levels of elastin, altered collagen types, and other biochemical changes which contribute to these enhanced properties. Examples of devices which would be improved by use of such tissue include heart valves, including percutaneous heart valves, and vascular grafts, patches and the like. Such enhanced materials can be sourced from specific populations of animals, such as neonatal calves, or in range-fed adult cattle, or can be fabricated or created from cell populations exhibiting Such properties. In one embodiment, glutaralde hyde-fixed neonatal pericardial tissue is used to create leaflets in a percutaneous heart Valve, and may be used without chemical fixation, with or without processes to remove residual cellular membranes, and utilized as a scaffold material for tissue engineering.

Biomimetic Scaffold Design for Tendon Tissue Engineering to Enhance Mechanoresponse and Tenogenesis

Islam A et al. 2017. Dissertation (Case Western Reserve University)
Application: Tendon

Tendon disorders are some of the most common musculoskeletal problems. Despite improvements in surgical techniques, revision of tendon tear surgery is significantly high. There is a recognized need for suitable bioscaffold that is porous enough for cell migration and proliferation, can promote tenogenic differentiation, and is simultaneously mechanically strong enough for tissue regeneration after implantation. The inability to incorporate high strength and high porosity in a structure has been one of the major barriers in the engineering of load-bearing tissue. There is a general lack of biofabrication methods that will provide macroporosity (0.5 mm or greater) with mechanical robustness in delicate protein-based biomaterials such as collagen. The current study developed a CAD/CAM based electrocompaction method to manufacture highly porous patterned scaffolds using pure collagen which has a potential to combine these characteristics. The lattice structures are made up of anisotropically aligned sheet like lattice structures. Therefore, the dissertation also develops a material model which considers stiffness as well as stiffness anisotropy (SA) to mimic two key features of natural tendon. Most natural tissues are substantially stronger along the load bearing direction than the direction transverse to the longer axis (such as tendons, muscle etc.). Notably, the cells of such tissues are elongated along the stiffer direction. There are not many biomaterials to emulate and study the effects of stiffness anisotropy on cellular response. In addition to the SA, extracellular matrix stiffness or mechanics also plays an important role to regulate cell morphology, proliferation, differentiation during regular and diseased states. There has been relatively little investigation on the roles of topographical factors (matrix anisotropy vs. matrix stiffness) in inducing tenogenic differentiation and the roles are still unknown. Elucidation of the mechanism of topographically induced tenogenesis in human MSCs will assist in optimizing the material to produce scaffolds for tendon repair and may provide insight into the differentiation mechanisms of MSCs and other stem cells in vivo, where type I collagen is the major substrate protein. Effect of SA and stiffness study demonstrated that stem cell fate is affected by not only the magnitude of stiffness but also by the directional anisotropy of the substrate stiffness. The study suggests that increasing stiffness anisotropy has a positive effect on tenogenesis. However, highly stiff substrate without SA can generate similar tenogenic differentiation as substrate with SA. This indicates, may be stiffness can overcome the effect of stiffness anisotropy on tenogenesis. Overall, SA has a positive effect on tenogenesis as anisotropy can be a key determinant in driving cell morphology and differentiation during development and maintenance of anistropically stiff tissues. In the stiffness study, a material profile was developed where the modulus can be changed from several hundred kPa, to single digit MPa, to hundred MPa. The tenoinductive collagen material model developed in the study can be used in the research and development of tissue engineering tendon repair constructs in future. The patterned lattice structure fabrication method demonstrated the capabilities of manufacturing mechanically robust ‘scaffolds with controlled porosity’. In this method, by changing the number of layers and shape of the structure, mechanical properties can be modulated for different tissue engineering application such as tendon, hernia, SUI, thoracic wall   reconstruction. Pure collagen based scaffolds have not been used in clinical mesh application due to their weak mechanics and usually collagen rich decellularized tissue based xenografts are used clinically when mechanical robustness is required. The fabrication method developed in this study generates pure collagen scaffold mesh within the mechanical strength range of decellularized tissue.

Bovine Pericardium Based Non-Cross Linked Collagen Matrix for Successful Root Coverage, a Clinical Study in Human

Schlee M et al. 2012. Head Face Med. 8:6
Application: Pericardium

The aim of this study was to clinically assess the capacity of a novel bovine pericardium based, non-cross linked collagen matrix in root coverage. 62 gingival recessions of Miller class I or II were treated. The matrix was adapted underneath a coronal repositioned split thickness flap. Clinical values were assessed at baseline and after six months. The mean recession in each patient was 2.2 mm at baseline. 6 Months after surgery 86.7% of the exposed root surfaces were covered. On average 0,3 mm of recession remained. The clinical attachment level changed from 3.5 ± 1.3 mm to 1,8 ( ± 0,7) mm during the observational time period. No statistically significant difference was found in the difference of probing depth. An increase in the width of gingiva was significant. With a baseline value of 1.5 ± 0.9 mm an improvement of 2.4 ± 0.8 mm after six month could be observed. 40 out of 62 recessions were considered a thin biotype at baseline. After 6 months all 62 sites were assessed thick. The results demonstrate the capacity of the bovine pericardium based non-cross linked collagen matrix for successful root coverage. This material was able to enhance gingival thickness and the width of keratinized gingiva. The percentage of root coverage achieved thereby is comparable to existing techniques. This method might contribute to an increase of patient's comfort and an enhanced aesthetical outcome.

Collagen as a Biological Glue for Cell Spraying in Wound Healing

Simpliciano C et al. TERMIS Americas, 2016 Annual Meeting
Application: Wound

In the US, about 6.5 million patients are treated for chronic wounds and burns, with a yearly financial burden of over $25 billion dollars. Worldwide, burns result in 265,000 deaths per year.  Currently, the gold standard for wound therapy is the use of either split- or full-thickness autologous skin grafts. Although widely used with positive outcomes, there are still disadvantages, including lack of healthy donor sites, risk of donor site infection, and the challenge of handling such delicate scaffolds. A more recent approach that simplifies the wound healing procedure is the application of autologous cells via a spray device.  Cell harvesting is considerably less invasive than skin harvesting, increasing the prospect of treating extensive wounds, even if donor sites are not available. The downside to cell spraying, however, is that cells have the tendency to fall off the wound site without a means to adhere. Collagen is a protein found naturally in the body, comprising about 75% of the skin. It is an essential component of the extracellular matrix and possesses wound-healing and hemostatic properties.  Poly(ethylene) glycol (PEG) is used regularly in tissue engineering and has a “sticky” quality that can be used to enhance the adhesive property of collagen. This purpose of this work is to design a collagen/PEG formulation for adhering cells onto a wound site and investigate whether the forces of spraying affects the survival of embedded cells.

Collagen Fibril Strain, Recruitment and Orientation for Pericardium Under Tension and the Effect of Cross Links

Kayed HR et al. 2015. RSC Adv. 5: 103703-103712
Application: Pericardium

The structural response of collagen fibrils in pericardium and other tissues when subjected to strain and the effect of cross linking on those structural changes are not well understood. Specifically, there is uncertainty about whether natural cross links of glycosaminoglycan (GAG) and synthetic cross links of glutaraldehyde have a mechanical function. Bovine pericardium was treated either with chondroitinase ABC to remove natural cross links or with glutaraldehyde to form synthetic cross links. The collagen fibril orientation index (OI) and D-spacing was measured on pericardium subjected to strain using synchrotron-based small angle X-ray scattering (SAXS). Under strain the collagen fibrils become much more oriented in the direction of the strain, with OI increasing from 0.25 to 0.89 in chondroitinase ABC-treated material, 0.22 to 0.93 in native material, and 0.22 to 0.77 in the glutaraldehyde-treated material. The proportion of fibrils that are recruited during stress varies from 36% in chondroitinase ABC-treated material, 12% in native material, to 45% in the glutaraldehyde-treated material. The increase in D-spacing shows the individual fibrils are strained in chondroitinase ABC-treated material by 2.4% on average or 4.6% for those in the direction of applied strain, in native material, 2.7% and 4.1%, respectively, and in the glutaraldehyde-treated material, 3.2% and 6.4%, respectively. Glutaraldehyde cross links are, therefore, shown to constrain the collagen fibrils and link them together mechanically. GAGs do not have such a marked mechanical effect; contrarily, the nature of internal structural responses to strain suggests that GAGs may have a lubricating rather than a binding effect.

Collagen Substrate Stiffness Anisotropy Affects Cellular Elongation, Nuclear Shape, and Stem Cell Fate Toward Anisotropic Tissue Lineage

Islam A et al. 2016. Adv Health Mat. 5(17): 2237-2247
Application: Tendon

Rigidity of substrates plays an important role in stem cell fate. Studies are commonly carried out on isotropically stiff substrate or substrates with unidirectional stiffness gradients. However, many native tissues are anisotropically stiff and it is unknown whether controlled presentation of stiff and compliant material axes on the same substrate governs cytoskeletal and nuclear morphology, as well as stem cell differentiation. In this study, electrocompacted collagen sheets are stretched to varying degrees to tune the stiffness anisotropy (SA) in the range of 1 to 8, resulting in stiff and compliant material axes orthogonal to each other. The cytoskeletal aspect ratio increased with increasing SA by about fourfold. Such elongation was absent on cellulose acetate replicas of aligned collagen surfaces indicating that the elongation was not driven by surface topography. Mesenchymal stem cells (MSCs) seeded on varying anisotropy sheets displayed a dose-dependent upregulation of tendon-related markers such as Mohawk and Scleraxis. After 21 d of culture, highly anisotropic sheets induced greater levels of production of type-I, type-III collagen, and thrombospondin-4. Therefore, SA has direct effects on MSC differentiation. These findings may also have ramifications of stem cell fate on other anisotropically stiff tissues, such as skeletal/cardiac muscles, ligaments, and bone.

Cross Linking and Fibril Alignment in Pericardium

Kayed HR et al. 2015. RSC Adv. 5: 3611-3618
Application: Pericardium

The influence of natural cross linking by glycosaminoglycan (GAG) on the structure of collagen in animal tissue is not well understood. Neither is the effect of synthetic cross linking on collagen structure well understood in glutaraldehyde treated collagenous tissue for medical implants and commercial leather. Bovine pericardium was treated with chondroitinase ABC to remove natural cross links or treated with glutaraldehyde to form synthetic cross links. The collagen fibril alignment was measured using synchrotron based small angle X-ray scattering (SAXS) and supported by atomic force microscopy (AFM) and histology. The alignment of the collagen fibrils is affected by the treatment. Untreated pericardium has an orientation index (OI) of 0.19 (0.06); the chondroitinase ABC treated material is similar with an OI of 0.21 (0.08); and the glutaraldehyde treated material is less aligned with an OI of 0.12 (0.05). This difference in alignment is also qualitatively observed in atomic force microscopy images. Crimp is not noticeably affected by treatment. It is proposed that glutaraldehyde cross linking functions to bind the collagen fibrils in a network of mixed orientation tending towards isotropic, whereas natural GAG cross links do not constrain the structure to quite such an extent.

Delivery of the Improved BMP-2-Advanced Plasmid DNA Within a Gene-Activated Scaffold Accelerates Mesenchymal Stem Cell Osteogenesis and Critical Size Defect Repair

Raftery RM et al. 2018. J Cont Rel. 283: 20-31
Application: Bone

Gene-activated scaffolds have been shown to induce controlled, sustained release of functional transgene both in vitro and in vivo. Bone morphogenetic proteins (BMPs) are potent mediators of osteogenesis however we found that the delivery of plasmid BMP-2 (pBMP-2) alone was not sufficient to enhance bone formation. Therefore, the aim of this study was to assess if the use of a series of modified BMP-2 plasmids could enhance the functionality of a pBMP-2 gene-activated scaffold and ultimately improve bone regeneration when implanted into a critical sized bone defect in vivo. A multi-cistronic plasmid encoding both BMP-2 and BMP-7 (BMP-2/7) was employed as was a BMP-2-Advanced plasmid containing a highly truncated intron sequence. With both plasmids, the highly efficient cytomegalovirus (CMV) promoter sequence was used. However, as there have been reports that the elongated factor 1-α promoter is more efficient, particularly in stem cells, a BMP-2-Advanced plasmid containing the EF1α promoter was also tested. Chitosan nanoparticles (CS) were used to deliver each plasmid to MSCs and induced transient up-regulation of BMP-2 protein expression, in turn significantly enhancing MSC-mediated osteogenesis when compared to untreated controls (p < 0.001). When incorporated into a bone mimicking collagen-hydroxyapatite scaffold, the BMP-2-Advanced plasmid, under the control of the CMV promotor, induced MSCs to produce approximately 2500 μg of calcium per scaffold, significantly higher (p < 0.001) than all other groups. Just 4 weeks post-implantation in vivo, this cell-free gene-activated scaffold induced significantly more bone tissue formation compared to a pBMP-2 gene-activated scaffold (p < 0.001) as indicated by microCT and histomorphometry. Immunohistochemistry revealed that the BMP-2-Advanced plasmid accelerated differentiation of osteoprogenitor cells to mature osteoblasts, thus causing rapid healing of the bone defects. This study confirms that optimising the plasmid construct can enhance the functionality of gene-activated scaffolds and translate to accelerated bone formation in a critical sized defect.

Ectopic Bone Formation in Rapidly Fabricated Acellular Injectable Dense Collagen-Bioglass Hybrid Scaffolds via Gel Aspiration-Ejection

Miri AK et al. 2016. Biomaterials. 85: 128-141
Application: Bone

Gel aspiration-ejection (GAE) has recently been introduced as an effective technique for the rapid production of injectable dense collagen (IDC) gel scaffolds with tunable collagen fibrillar densities (CFDs) and microstructures. Herein, a GAE system was applied for the advanced production and delivery of IDC and IDC-Bioglass(®) (IDC-BG) hybrid gel scaffolds for potential bone tissue engineering applications. The efficacy of GAE in generating mineralizable IDC-BG gels (from an initial 75-25 collagen-BG ratio) produced through needle gauge numbers 8G (3.4 mm diameter and 6 wt% CFD) and 14G (1.6 mm diameter and 14 wt% CFD) was investigated. Second harmonic generation (SHG) imaging of as-made gels revealed an increase in collagen fibril alignment with needle gauge number. In vitro mineralization of IDC-BG gels was confirmed where carbonated hydroxyapatite was detected as early as day 1 in simulated body fluid, which progressively increased up to day 14. In vivo mineralization of, and host response to, acellular IDC and IDC-BG gel scaffolds were further investigated following subcutaneous injection in adult rats. Mineralization, neovascularization and cell infiltration into the scaffolds was enhanced by the addition of BG and at day 21 post injection, there was evidence of remodeling of granulation tissue into woven bone-like tissue in IDC-BG. SHG imaging of explanted scaffolds indicated collagen fibril remodeling through cell infiltration and mineralization over time. In sum, the results suggest that IDC-BG hybrid gels have osteoinductive properties and potentially offer a novel therapeutic approach for procedures requiring the injectable delivery of a malleable and dynamic bone graft that mineralizes under physiological conditions.

Effect of Actuating Cell Source on Locomotion of Organic Living Machines with Electrocompacted Collagen Skeletons

Webster VA et al. 2016. Bioins & Biomim. 11(3): 1-13
Application: Cardiac Muscle

In robotics, there is a need for small scale, compliant actuators for use in medical applications or minimally invasive environmental monitoring. Biohybrid devices offer one solution to this need by using muscle cells to actuate compliant scaffolds. Such devices typically use biocompatible synthetic polymers as compliant scaffolds, which require additional processing steps to promote cellular alignment and attachment. Instead, electrocompacted and aligned collagen (ELAC) can be used as a completely organic scaffold, requiring no additional processing steps, with alignment being innately promoted by the topography. Locomotive living machines have been fabricated in this study using ELAC scaffolds. Devices have been produced using either primary cardiomyocytes or primary skeletal muscle cells isolated from chick embryos as actuators. When tested under the same conditions, skeletal muscle cell powered devices were approximately an order of magnitude faster, having a mean velocity of 77.6 ± 86.4 μm min–1, compared to 9.34 ± 6.69 μm min–1 for cardiomyocyte powered devices. In conclusion, completely organic living machines have been fabricated using electrocompacted collagen skeletons, and it was found that skeletal muscle powered devices were significantly faster than cardiomyocyte powered devices.

Effects of Substrate Stiffness on the Tenoinduction of Human Mesenchymal Stem Cells

Islam A et al. 2017. Acta Biomater. 58:244-253
Application: Tendon

Extracellular matrix modulus plays an important role in regulating cell morphology, proliferation and differentiation during regular and diseased states. Although the effects of substrate topography and modulus on MSC differentiation are well known with respect to osteogenesis and adipogenesis, there has been relatively little investigation on the effects of this phenomenon on tenogenesis. Furthermore, relative roles of topographical factors (matrix alignment vs. matrix modulus) in inducing tenogenic differentiation is not well understood. In this study we investigated the effects of modulus and topographical alignment of type I collagen substrate on
tendon differentiation. Type I collagen sheet substrates with random topographical alignment were fabricated with their moduli tuned in the range of 0.1, 1, 10 and 100 MPa by using electrocompaction and controlled crosslinking. In one of the groups, topographical alignment was introduced at 10 MPa stiffness, by controlled unidirectional stretching of the sheet. RT-PCR, immunohistochemistry and immunofluorescence results showed that mimicking the tendon topography, i.e. increasing the substrate modulus as well as alignment increased the tenogenic differentiation. Higher substrate modulus increased the expression of COLI, COLIII, COMP and TSP-4 about 2–3 fold and increased the production of COLI, COLIII and TSP-4 about 2–4 fold. Substrate alignment up regulated COLIII and COMP expression by 2 fold. Therefore, the tenoinductive collagen material model developed in this study can be used in the research and development of tissue engineering tendon repair constructs in future.

Electrospin Silk-Collagen Scaffolds and BMP-14 for Ligament and Tendon Repair and Regeneration

Maghdouri-White Y et al. 2018. Biomed Phys & Eng Exp. https://orcid.org/0000-0002-2010-2165
Application: Tendon

Regenerative ligament and tendon repair scaffolds have been highly researched, yet few match the mechanical properties of native tissue, while fewer drugs have been explored for enhancing cell infiltration into the damaged tissue. Here a nanofiber scaffold of silk fibroin (SF)–collagen blend is explored as a biologically enhanced matrix, along with a therapeutic agent (bone morphogenetic protein-13, [BMP-13]) for connective tissue regeneration. SF and collagen were blended and electrospun to form fibrous scaffolds with 1.15 ± 0.08 μm diameter fibers. These scaffolds were crosslinked with either methanol or ethanol. Crosslinking with methanol resulted in significantly higher mechanical strength compared to ethanol treated scaffolds (2.92 ± 0.21 MPa versus 1.13 ± 0.08 MPa, respectively). Adipose-derived stem cells showed robust cell attachment and proliferation on SF–collagen scaffolds, with confocal imaging suggesting cellular alignment and spreading. BMP-13 growth factor is further shown to promote cell migration into SF–collagen scaffolds. In all, electrospun SF with telocollagen produces a regenerative matrix with enhanced tensile strength. BMP-13 improves cellular infiltration into electrospun SF–collagen scaffolds and may prove a potent chemotactic agent for enhancing ligament and tendon repair.

Evaluation of a Collagen-Coated, Resporbable Fiber Scaffold Loaded with a Peptide Basic Fibroblast Growth Factor Mimetic in a Sheep Model of Rotator Cuff Repair

Peterson DR et al. 2015. J Shoulder Elbow Surg. 24(11) 1764-1773
Application: Tendon

A new scaffold design combined with a peptide growth factor was tested prospectively for safety and for improved tendon healing in sheep. The infraspinatus tendon was detached and then surgically repaired to the humerus using sutures and anchors in 50 adult sheep. The repairs in 40 of these sheep were reinforced with a scaffold containing F2A, a peptide mimetic of basic fibroblast growth factor. The sheep were examined after 8 or 26 weeks with magnetic resonance imaging, full necropsy, and histopathologic analysis. A second cohort of 30 sheep underwent surgical repair—20 with scaffolds containing F2A. The 30 shoulders were tested mechanically after 8 weeks. The scaffold and F2A showed no toxicity. Scaffold-repaired tendons were 31% thicker than surgically repaired controls (P = .037) at 8 weeks. There was more new bone formed at the tendon footprint in sheep treated with F2A. Surgically repaired tendons delaminated from the humerus across 14% of the footprint area. The extent of delamination decreased to 1.3% with increasing doses of F2A (P = .004). More of the repair tissue at the footprint was tendon-like in the peptide-treated sheep. On mechanical testing, only 7 shoulders tore at the repair site. The repairs in the other 23 shoulders were already stronger than the midsubstance tendon at 8 weeks. The new scaffold and peptide safely improved tendon healing.

Evaluation of Fibroblast Behavior on a Collagen-Based Spray Adhesive

Simpliciano C et al. Society for Biomaterials, 2017 Annual Meeting
Application: Wound

The ongoing search for a suitable surgical adhesive is fueled by deficiencies of current products such as mechanical properties, bonding strengths and curing times. An adhesive in a sprayable form has much appeal for ease of application. Collagen has shown great potential as an adhesive for its biocompatibility, blood absorption and hemostatic properties1. The addition of an alcohol results in an esterification reaction with collagen carboxylic groups, producing methylated collagen, which is soluble, and therefore sprayable, at physiological pH. Introducing a multifunctional polymer such as polyethylene glycol (PEG) cross-links collagen, forming a bond between two surfaces. We aim to show the biocompatibility of a potential sprayable adhesive composed of gelled methylated collagen with succinimidyl glutarate (SG) and thiol (SH) PEGs by observing the proliferation and viability of fibroblast cells entrapped within the collagen/PEG matrix.

Evaluation of the Immunomodulatory Effects of All-Trans Retinoic Acid Solid Lipid Nanoparticles and Human Mesenchymal Stem Cells in an A549 Epithelial Cell Line Model

Payne CM et al. 2019. Pharm Res. 36:50
Application: Cell Delivery, Application: Drug Delivery

Purpose
To investigate two potential strategies aimed at targeting the inflammatory pathogenesis of COPD: a small molecule, all trans retinoic acid (atRA) and human mesenchymal stem cells (hMSCs).
Methods
atRA was formulated into solid lipid nanoparticles (SLNs) via the emulsification-ultrasonication method, and these SLNs were characterised physicochemically. Assessment of the immunomodulatory effects of atRA-SLNs on A549 cells in vitro was determined using ELISA. hMSCs were suspended in a previously developed methylcellulose, collagen and beta-glycerophosphate hydrogel prior to investigating their immunomodulatory effects in vitro.
Results
SLNs provided significant encapsulation of atRA and also sustained its release over 72 h. A549 cells were viable following the addition of atRA SLNs and showed a reduction in IL-6 and IL-8 levels. A549 cells also remained viable following addition of the hMSC/hydrogel formulation – however, this formulation resulted in increased levels of IL-6 and IL-8, indicating a potentially pro-inflammatory effect.
Conclusion
Both atRA SLNs and hMSCs show potential for modulating the environment in inflammatory disease, though through different mechanisms and leading to different outcomes – despite both being explored as strategies for use in inflammatory disease. atRA shows promise by acting in a directly anti-inflammatory manner, whereas further research into the exact mechanisms and behaviours of hMSCs in inflammatory diseases is required.

Flow Simulation of a Natural Polymer in a Syringe-Needle Delivery Device

Syntouka IM et al. 2018. ECCM6 & ECFD 7
Application: Cell Delivery

Neurodegenerative diseases, such as Parkinson’s disease, affect a large number of the erderly population and still remain untreated. In recent years, cell therapy has emerged as a promising therapeutic strategy. To increase cell viability, biomaterials are often used as scaffolds and facilitate cell deposition, through injection, to the site of interest. However, fluid forces acting on the cells during injection may lead to their disruption or death. This study aims to develop a novel device for the delivery of a cell-embedded, in situ forming, collagen hydrogel. A preliminary simulation study on constricted channels representing the syringe was performed to gain insight into the effect of needle diameter and syringe geometry. Straight needles emanating co-axially from syringes of various geometries were computationally modelled in the two-dimensional space, using OpenFOAM R. The natural collagen solution was modelled as a continuum medium, without cells, and the flow was assumed incompressible, with non-Newtonian fluid constitutive behaviour. The effects of needle diameter and syringe geometry on velocity and shear stresses were examined. The results highlight the importance of geometric characteristics on the design of new cell delivery devices. If cells pass from the syringe barrel to the needle, the pressure drop and the increased velocity could damage them. This is more likely to occur using higher Gauge needles. Further analysis is required including simulations of cells during injection and analysis of their deformation.

Formulation of Poly(Ethylene Glycol) Hydrogens for Drug Delivery

Lieberthal T, Kao WH. 2016. Polymeric Drug Delivery Techniques. Sigma-Aldrich Spotlight. 31-35
Application: Drug Delivery

Although described as a “bioinert” drug delivery platform, PEG hydrogels can also incorporate bioactive materials, such as extracellular matrix amino acid motifs or macromolecules such as collagen. Incorporation of biological macromolecules facilitates integration into the native tissue environment due to cell-integrin recognition of extracellular matrix components such as laminin, fibronectin, collagen, and hyaluronic acid. This may be important for drug delivery applications where degradation of the hydrogel, and therefore drug release, should be mediated by the host microenvironment. Control of degradation is, therefore, critically dependent on the composition of the hydrogel and the sensitivity of the degradable sequences. 

Heparinized Collagen Sutures for Sustained Delivery of PDGF-BB: Delivery Profile and Effects on Tendon-Derived Cells In-Vitro

Younesi M et al. 2017. Acta Bio. 41: 100-109
Application: Tendon

Suturing is the standard of repair for lacerated flexor tendons. Past studies focused on delivering growth factors to the repair site by incorporating growth factors to nylon sutures which are commonly used in the repair procedure. However, conjugation of growth factors to nylon or other synthetic sutures is not straightforward. Collagen holds promise as a suture material by way of providing chemical sites for conjugation of growth factors. On the other hand, collagen also needs to be reconstituted as a mechanically robust thread that can be sutured. In this study, we reconstituted collagen solutions as suturable collagen threads by using linear electrochemical compaction. Prolonged release of PDGF-BB (Platelet derived growth factor-BB) was achieved by covalent bonding of heparin to the collagen sutures. Tensile mechanical tests of collagen sutures before and after chemical modification indicated that the strength of sutures following chemical conjugation stages was not compromised. Strength of lacerated tendons sutured with epitendinous collagen sutures (11.2 ± 0.7 N) converged to that of the standard nylon suture (14.9 ± 2.9 N). Heparin conjugation of collagen sutures didn’t affect viability and proliferation of tendon-derived cells and prolonged the PDGF-BB release up to 15 days. Proliferation of cells seeded on PDGF-BB incorporated collagen sutures was about 50% greater than those seeded on plain collagen sutures. Collagen that is released to the media by the cells increased by 120% under the effects of PDGF-BB and collagen production by cells was detectable by histology as of day 21. Addition of PDGF-BB to collagen sutures resulted in a moderate decline in the expression of the tendon-associated markers scleraxis, collagen I, tenomodulin, and COMP; however, expression levels were still greater than the cells seeded on collagen gel. The data indicate that the effects of PDGF-BB on tendon-derived cells mainly occur through increased cell proliferation and that longer term studies are needed to confirm whether this proliferation is outweighs the moderate reduction in the expression of tendon-associated genes.

Immediate Production of a Tubular Dense Collagen Construct with Bioinspired Mechanical Properties

Ghezzi CE. 2012. Acta Bio. 8: 1813-1825
Application: General Collagen

The intrinsic complexity of tissues and organs demands tissue engineering approaches that extend beyond planar constructs currently in clinical use. However, the engineering of cylindrical or tubular tissue constructs with a hollow lumen presents significant challenges arising from geometrical and architectural considerations required to tailor biomaterials for tissue and organ repair. Type I collagen is an ideal scaffolding material due to its outstanding biocompatibility and high processability. However, the highly hydrated nature of collagen hydrogels results in their lack of mechanical properties and instability, as well as extensive cell-mediated contraction, which must be overcome to achieve process control. Herein, tubular dense collagen constructs (TDCCs) were produced simply and rapidly (in less than 1h) by circumferentially wrapping plastically compressed dense collagen gel sheets around a cylindrical support. The effects of collagen source, i.e. rat-tail tendon and bovine dermis-derived acid solubilized collagen, and concentration on TDCC properties were investigated through morphological, mechanical and chemical characterizations. Both tensile strength and apparent modulus correlated strongly with physiologically relevant collagen gel fibrillar densities. The clinical potential of TDCC as a tubular tissue substitute was demonstrated mechanically, through circumferential tensile properties, theoretical burst pressure, which ranged from 1225 to 1574 mm Hg, compliance values of between 8.3% to 14.2% per 100mm Hg and suture retention strength in the range of 116-151 grams-force, which were compatible with surgical procedures. Moreover, NIH/3T3 fibroblast viability and uniform distribution within the construct wall were confirmed up to day 7 in culture. TDCCs with fibrillar densities equivalent to native tissues can be readily engineered in various dimensions with tunable morphological and mechanical properties, which can be easily handled for use as tissue models and adapted to clinical needs.

Influence of Telopeptides on the Structural and Physical Properties of Polymeric and Monomeric Acid-Soluble Type I Collagen

Holmes R et al. 2017. Mater Sci & Eng. C. 77:823-827
Application: General Collagen

<p>Currently two factors hinder the use of collagen as building block of regenerative devices: the limited mechanical strength in aqueous environment, and potential antigenicity. Polymeric collagen is naturally found in the cross-linked state and is mechanically tougher than the monomeric, acid-soluble collagen ex vivo. The antigenicity of collagen, on the other hand, is mainly ascribed to inter-species variations in amino acid sequences of the non-helical terminal telopeptides. These telopeptides can be removed through enzymatic treatment to produce atelocollagen, although the effect of this cleavage on triple helix organization, amino acidic composition and thermal properties is often disregarded. Here, we compare the structural, chemical and physical properties of polymeric and monomeric type I collagen with and without telopeptides, in an effort to elucidate the influence of either mature covalent crosslinks or telopeptides. Circular dichroism (CD) was used to examine the triple helical conformation and quantify the denaturation temperature (Td) of both monomeric collagen (36.5 °C) and monomeric atelocollagen (35.5 °C). CD measurements were combined with differential scanning calorimetry (DSC) in order to gain insight into the triple helix-to-coil thermal transition and shrinkage temperature (Ts) of polymeric atelo collagen (44.8 °C), polymeric collagen (62.7 °C), monomeric atelo collagen (51.4 °C) and monomeric collagen (66.5 °C). Structural and thermal analysis was combined with high pressure liquid chromatography (HPLC) to determine the content of specific collagen amino acidic residues used as markers for the presence of telopeptides and mature crosslinks. Hydroxylamine was used as the marker for polymeric collagen, and had a total content of 9.66% for both polymeric and polymeric atelo collagen; tyrosine was used as the marker for telopeptide cleavage, was expressed as 0.526% of the content of polymeric collagen and the partially-reduced content of 0.39% for atelocollagen.</p>

Labeling of Collagen Type I Templates with a Naturally Derived Contrast Agent for Noninvasive MR Imaging in Soft Tissue Engineering

Janke HP et al. 2018. Adv Health Mat. https://doi.org/10.1002/adhm.201800605
Application: General Collagen

In vivo monitoring of tissue‐engineered constructs is important to assess their integrity, remodeling, and degradation. However, this is challenging when the contrast with neighboring tissues is low, necessitating labeling with contrast agents (CAs), but current CAs have limitations (i.e., toxicity, negative contrast, label instability, and/or inappropriate size). Therefore, a naturally derived hemin‐L‐lysine (HL) complex is used as a potential CA to label collagen‐based templates for magnetic resonance imaging (MRI). Labeling does not change the basic characteristics of the collagen templates. When hybrid templates composed of collagen type I reinforced with degradable polymers are subcutaneously implanted in mice, longitudinal visualization by MRI is possible with good contrast and in correlation with template remodeling. In contrast, unlabeled collagen templates are hardly detectable and the fate of these templates cannot be monitored by MRI. Interestingly, tissue remodeling and vascularization are enhanced within HL‐labeled templates. Thus, HL labeling is presented as a promising universal imaging marker to label tissue‐engineered implants for MRI, which additionally seems to accelerate tissue regeneration.

Nerve Growth Factor Released from Collagen Scaffolds Protects Axotomized Cholinergic Neurons of the Basal Nucleus of Meynert in Organotypic Brain Slices

Foidl BM et al. 2017. J of Neu Meth. https://doi.org/10.1016/j.jneumeth.2017.12.003.
Application: Drug Delivery

Background: Alzheimer´s disease is accompanied by cell death of cholinergic neurons, resulting in cognitive impairment and memory loss. Nerve growth factor (NGF) is the most potent protein to support survival of cholinergic neurons. New Method: Organotypic brain slices of the basal nucleus of Meynert (nBM) are a valuable tool to study cell death of axotomized cholinergic neurons, as well as protective effects of NGF added into the medium. The aim of the present study is to use collagen scaffolds crosslinked with polyethylineglycole and load with NGF to target delivery of NGF to organotypic nBM brain slices. Results: Collagen scaffolds (visualized by incorporating AlexaFluor 488 antibodies) slowly degraded when applied onto organotypic brain slices within 2 weeks in culture. GFAP reactive astrocytes and Iba1+ microglia became visible around the collagen scaffolds 7 days after incubation, showing reactive gliosis. Cholinergic neurons of the nBM survived (201±21, n=8) when incubated with 100 ng/ml NGF in the medium compared to NGF-free medium (69±12, n=7). Collagen scaffolds loaded with NGF (1 ng/2 μl scaffold) significantly rescued cholinergic cell death in the nBM brain slices (175±12, n=10), which was counteracted by an anti-NGF antibody (77±5, n=5). Comparison with existing Methods: The combination of coronal brain slices with biomaterial is a novel and potent tool to selectively study neuroprotective effects. Conclusions: Collagen scaffolds loaded with low amounts of a protein/drug of interest can be easily applied directly onto organotypic brain slices, allowing slow targeted release of a protective molecule. Such an approach is highly useful to optimize CollScaff for further in vivo applications.

On-Demand Release of Hydrosoluble Drugs from a Paramagnetic Porous Collagen-Based Scaffold

Bettini S et al. 2016. Chem Eur J. 22: 1-9
Application: Drug Delivery

The design of a collagen scaffold containing iron oxide nanostructures capped by a TiO2 (anatase) layer is reported. The TiO2 shell is proposed to perform a dual role: 1) as an innovative and biocompatible cross-linker agent, providing binding sites to the protein moiety, through the well-known TiO2 chemical affinity towards carboxyl groups, and 2) as a protective surface layer for the paramagnetic core against oxidation. Simultaneously, the presence of the nanostructures confers to the collagen gel sensitivity to an external stimulus; that is, the application of a magnetic field. The hybrid biomaterial was demonstrated to be nontoxic and is proposed as a smart scaffold for the release of bioactive compounds on demand. The tunable release of a model protein (myoglobin) upon application of a magnetic field was investigated. Myoglobin was loaded in the microporous material and discharge was induced by consecutive magnet applications, leading to release of the protein with high spatio–temporal and dosage control.

Pneumatospinning of Collagen Microfibers from Benign Solvents

Polk S et al. 2018. Biofabrication. https://doi.org/10.1088/1758-5090/aad7d0
Application: General Collagen

Introduction. Current collagen fiber manufacturing methods for biomedical applications, such as electrospinning and extrusion, have had limited success in clinical translation, partially due to scalability, cost, and complexity challenges. Here we explore an alternative, simplified and scalable collagen fiber formation method, termed 'pneumatospinning,' to generate submicron collagen fibers from benign solvents. Methods and results. Clinical grade type I atelocollagen from calf corium was electrospun or pneumatospun as sheets of aligned and isotropic fibrous scaffolds. Following crosslinking with genipin, the collagen scaffolds were stable in media for over a month. Pneumatospun collagen samples were characterized using Fourier-transform infrared spectroscopy, circular dichroism, mechanical testing, and scanning electron microscopy showed consistent fiber size and no deleterious chemical changes to the collagen were detected. Pneumatospun collagen had significantly higher tensile strength relative to electrospun collagen, with both processed from acetic acid. Stem cells cultured on pneumatospun collagen showed robust cell attachment and high cytocompatibility. Using DMSO as a solvent, collagen was further co-pneumatospun with poly(d,l-lactide) to produce a blended microfibrous biomaterial. Conclusions. Collagen microfibers are shown for the first time to be formed using pneumatospinning, which can be collected as anisotropic or isotropic fibrous grafts. Pneumatospun collagen can be made with higher output, lower cost and less complexity relative to electrospinning. As a robust and rapid method of collagen microfiber synthesis, this manufacturing method has many applications in medical device manufacturing, including those benefiting from anisotropic microstructures, such as ligament, tendon and nerve repair, or for applying microfibrous collagen-based coatings to other materials.

Protease-Sensitive Atelocollagen Hydrogels Promote Healing in a Diabetic Wound Model

Tronci G et al. 2016. J Mater Chem B. 4: 7249-7258
Application: Wound

The design of exudate-managing wound dressings is an established route to accelerated healing, although such design remains a challenge from material and manufacturing standpoints. Aiming towards the clinical translation of knowledge gained in vitro with highly-swollen rat tail collagen hydrogels, this study investigated the healing capability in a diabetic mouse wound model of telopeptide-free, protease-inhibiting collagen networks. 4-Vinylbenzylation and UV irradiation of type I atelocollagen (AC) led to hydrogel networks with chemical and macroscopic properties comparable to previous collagen analogues, attributable to similar lysine content and dichroic properties. After 4 days in vitro, hydrogels induced nearly 50 RFU% reduction in matrix metalloproteinase (MMP)-9 activity, whilst showing less than 20 wt% mass loss. After 20 days in vivo, dry networks promoted 99% closure of 10 × 10 mm full thickness wounds and accelerated neo-dermal tissue formation compared to Mepilex®. This collagen system can be equipped with multiple, customizable properties and functions key to personalized chronic wound care.

Repair of Large Osteochondritis Dissecans Lesions Using a Novel Multilayered Tissue Engineered Construct in an Equine Athlete

Stack JD et al. 2016. J of TERM.
Application: Bone/Cartilage

Osteochondral lesions resulting from osteochondritis dissecans are problematic to treat and present a significant challenge for clinicians. The aims of this study were to investigate the use of a scaffold-assisted microfracture approach, employing a novel, multilayered, collagen-based, osteochondral graft substitute in the treatment of severe osteochondritis dissecans of both lateral femoral trochlear ridges in an equine athlete, and to assess the potential of this novel scaffold to enhance repair of the osteochondral unit. A 15 month-old female filly presented with large osteochondritis dissecans lesions involving both femoral lateral trochlear ridges. After routine arthroscopic debridement and microfracture of the subchondral bone, multilayered osteochondral defect repair scaffolds were implanted into the fragmentation beds in both left and right femoropatellar joints via mini-arthrotomes. Exploratory arthroscopy 5 months postimplantation revealed smooth cartilaginous repair tissue, contiguous with the adjacent cartilage, covering the defect. At 22-month follow up, the filly had no signs of lameness and was exercising at her intended level. Radiographically, although still slightly flattened, the femoral trochlear ridges were smooth, with no evidence of osteoarthritis. Ultrasonographically, the defects were filled with bone and covered with an overlying cartilaginous layer, with the trochlear ridge contour almost entirely restored. This report demonstrates the effective clinical use of this novel, multilayered, osteochondral defect repair scaffold in the treatment of osteochondritis dissecans of an equine athlete. The successful repair achieved here using this novel scaffold in an equine patient with large bilateral lesions shows the potential for clinical translation in the treatment of human patients presenting with osteochondral defects.

Scaffold Design for Artificial Tissue With Bone Marrow Stem Cells

Noreikaite A et al. 2017. Medicina. http://dx.doi.org/10.1016/j.medici.2017.07.001
Application: General Collagen

Objective: The aim of this study was to test polymeric materials (collagen, fibrin, polyimide film, and polylactic acid) for single- and multi-layer scaffold formation. Materials and methods: In our study, we used rabbit bone marrow stem cells (rBMSCs) and human mesenchymal stem cells (hMSCs) with materials of a different origin for the formation of an artificial scaffold, such as a collagen scaffold, fibrin scaffold produced from clotted rabbit plasma, electrospun poly(lactic acid) (PLA) mats, polyimide film (PI), and the combination of the latter two. Cell imaging was performed 3–14 days after cell cultivation in the scaffolds. Time-lapse imaging was used to determine hMSC mobility on the PI film. Results: Cell incorporation in collagen and clotted fibrin scaffolds was evaluated after 2-week cultivation in vitro. Histological analysis showed that cells penetrated only external layers of the collagen scaffold, while the fibrin clot was populated with rBMSCs through the entire scaffold thickness. As well, cell behavior on the laser micro-structured PI film was analyzed. The mobility of hMSCs on the smooth PI film and the micro-machined surface was 20 ± 2 mm/h and 18 ± 4 mm/h, respectively. After 3-day cultivation, hMSCs were capable of spreading through the whole 100 ± 10 mm-thick layer of the electrospun PLA scaffold and demonstrated that the multilayer scaffold composed of PI and PLA materials ensured a suitable environment for cell growth. Conclusions: The obtained results suggest that electrospinning technology and femtosecond laser micro-structuring could be employed for the development of multi-layer scaffolds. Different biopolymers, such as PLA, fibrin, and collagen, could be used as appropriate environments for cell inhabitation and as an inner layer of the multi-layer scaffold. PI could be suitable as a barrier blocking cell migration from the scaffold. However, additional studies are needed to determine optimal parameters of inner and outer scaffold layers.

Structurally Graduated Collagen Scaffolds Applied to the Ex Vivo Generation of Platelets from Human Pluripotent Stem Cell-Derived Megakaryocytes: Enhancing Production and Purity

Shepherd JH et al. 2018. Biomaterials. doi: 10.1016/j.biomaterials.2018.08.019.
Application: Cell Delivery

Platelet transfusions are a key treatment option for a range of life threatening conditions including cancer, chemotherapy and surgery. Efficient ex vivo systems to generate donor independent platelets in clinically relevant numbers could provide a useful substitute. Large quantities of megakaryocytes (MKs) can be produced from human pluripotent stem cells, but in 2D culture the ratio of platelets harvested from MK cells has been limited and restricts production rate. The development of biomaterial cell supports that replicate vital hematopoietic micro-environment cues are one strategy that may increase in vitro platelet production rates from iPS derived Megakaryocyte cells. In this paper, we present the results obtained generating, simulating and using a novel structurally-graded collagen scaffold within a flow bioreactor system seeded with programmed stem cells. Theoretical analysis of porosity using micro-computed tomography analysis and synthetic micro-particle filtration provided a predictive tool to tailor cell distribution throughout the material. When used with MK programmed stem cells the graded scaffolds influenced cell location while maintaining the ability to continuously release metabolically active CD41 + CD42 + functional platelets. This scaffold design and novel fabrication technique offers a significant advance in understanding the influence of scaffold architectures on cell seeding, retention and platelet production.

Supercritical Carbon Dioxide Decellularized Pericardium: Mechanical and Structural Characterization for Applications in Cardio-Thoracic Surgery

Halfwerk FR et al. 2018. J of Mech Behav of Biomed Mat. 77: 400-407
Application: Pericardium

Introduction: Many biomaterials are used in cardio-thoracic surgery with good short-term results. However, calcification, dehiscence, and formation of scar tissue are reported. The aim of this research is to characterise decellularised pericardium after supercritical carbon dioxide (scCO2) processing as an alternative biological material for uses in cardio-thoracic surgery. Methods: Porcine and bovine pericardium were decellularised using scCO2. Mechanical properties such as tensile strength, elastic modulus, fracture toughness and suture retention strength were determined. Ultrastructure was visualised using Scanning Electron Microscopy. Water uptake and swelling was experimentally determined. Commercially available glutaraldehyde treated bovine pericardium was used as gold standard for comparison. Results: scCO2 decellularised porcine (and bovine pericardium) maintained their tensile strength compared to untreated native pericardium (13.3 ± 2.4 MPa vs 14.0 ± 4.1 MPa, p = 0.73). Tensile strength of glutaraldehyde treated pericardium was significantly higher compared to untreated pericardium (19.4 ± 7.3 MPa vs 10.2 ± 2.2 MPa, p = 0.02). Suture retention strength of scCO2 treated pericardium was significantly higher than glutaraldehyde treated pericardium (p = 0.01). We found no anisotropy of scCO2 or glutaraldehyde treated pericardium based on a trouser tear test. Ultrastructure was uncompromised in scCO2 treated pericardium, while glutaraldehyde treated pericardium showed deterioration of extracellular matrix. Conclusion: scCO2 processing preserves initial mechanical and structural properties of porcine and bovine pericardium, while glutaraldehyde processing damages the extracellular matrix of bovine pericardium. Decellularisation of tissue using scCO2 might give long-term solutions for cardio-thoracic surgery without compromising initial good mechanical properties.

Synthesis and Fabrication of Nanocomposite Fibers of Collagen-Cellulose Nanocrystals by Coelectrocompaction

Cudjoe E et al. 2017. Biomac. 18(4): 1259-1267
Application: General Collagen

An electrochemical process has been used to compact cellulose nanocrystals (CNC) and access aligned micron-sized CNC fibers. Placing a current across aqueous solutions of carboxylic acid functionalized CNCs (t-CNC–COOH) or carboxylic acid/primary amine functionalized CNCs (t-CNC–COOH-NH2) creates a pH gradient between the electrodes, which results in the migration and concentration of the CNC fibers at their isoelectric point. By matching the carboxylic acid/amine ratio of CNCs and collagen (ca. 30:70 carboxylic acid:amine ratio), it is possible to coelectrocompact both nanofibers and access aligned nanocomposite fibers. t-CNC–COOH-NH2/collagen fibers showed a maximum increase in mechanical properties at 5 wt % of t-CNC–COOH-NH2. Compared to collagen/CNC films which have no alignment in the plane of the films, the tensile properties of the aligned fibers show a significant enhancement in the wet mechanical properties (40 MPa vs 230 MPa) for the 5 wt % of t-CNC–COOH-NH2/collagen films and fiber, respectively.

The Effect of Different Collagen Modifications for Titanium and Titanium Nitrite Surfaces on Functions of Gingival Fibroblasts

Ritz U et al. 2016. Clin Oral Invest. 21: 1
Application: Dental

Targeted modifications of the bulk implant surfaces using bioactive agents provide a promising tool for improvement of the long-term bony and soft tissue integration of dental implants. In this study, we assessed the cellular responses of primary human gingival fibroblasts (HGF) to different surface modifications of titanium (Ti) and titanium nitride (TiN) alloys with type I collagen or cyclic-RGDfK-peptide in order to define a modification improving long-term implants in dental medicine.

Therapeutic Efficacy of Glial Cell-Derived Neurotrophic Factor Loaded Collagen Scaffolds in Ex Vivo Organotypic Brain Slice Parkinson's Disease Models

Ucar B and Humpel C. 2019. Brain Res Bul. 149:86-95
Application: Drug Delivery

Glial cell line-derived neurotrophic factor (GDNF) is a potent trophic factor that supports the survival of dopaminergic neurons of the substantia nigra (SN), which degenerate in Parkinson’s disease (PD). The application of GDNF to the brain is challenging but biomaterials such as collagen can present novel strategies to target therapeutics to the brain. In this study, we assess the efficacy of collagen scaffolds loaded with GDNF on dopaminergic neuronal survival in organotypic ex vivo slices: axotomy, rotenone, and 6-hydroxydopamine (6-OHDA) models. Coronal (150 μm) mesencephalon brain slices were prepared from postnatal day 9–11 mice. In these slices 424 ± 32 and 158 ± 26 dopamine neurons were found in SN and ventral tegmental area, respectively. Collagen was crosslinked with poly(ethylene glycol), loaded with GDNF and drops of 2 μl collagen scaffold containing 10 ng GDNF were directly placed onto organotypic brain slices. GDNF released from collagen scaffolds significantly protected dopaminergic SN neurons against axotomy and rotenone (50 nM, 14 days) induced cell death and showed a tendency of neuroprotection in 6-OHDA (5 mM, 10 min) lesions. In the axotomy model GDNF (100 ng/ml in medium) markedly enhanced tyrosine hydroxylase (TH) expression, which was verified by Western Blot and qRT-PCR. Our results indicate that this approach has the potential to be used as an injectable hydrogel system to address the need of targeted long-term growth factor delivery for slowing or stopping disease progression in the future.

Towards an Effective, Needle-Based Delivery Device for Parkinson's Disease: A Simulation Study on the Impact of Needle Diameter

Syntouka et al. 2018.8th World Congress of Biomechanics
Application: Cell Delivery

Recently, several therapies have emerged for Parkinson’s disease, a challenging neurodegenerative disorder. However, clinical translation is restricted, partially due to limitations in delivering therapeutics to the Central Nervous System (CNS) which cannot be reached by systemic administration. An alternative method, that bypasses the blood brain barrier and offers high-concentrated dposition in the diseased region, is intrastriatal delivery of a cell-loaded in situ forming collagen hydrogel. However, this strategy has disadvantages, including neuroimmune response and haemorrhage. To minimize these responses, an optimised medical device should be designed. Of main consideration is the volume dispensed and the needle dimensions. Current approaches use 18-20-Guaged diameter needles and multiple cranial penetrations. Additionally, fluid forces acting on cells may lead to cell disruption and death. This study aims to develop a novel device for the effective delivery of a cell-loaded in situ forming collagen hydrogel to the CNS. A simulation study on constricted channels representing the needle was performed to gain insight into the optimal needle diameter.

Translating the Role of Osteogenic-Angiogenic Coupling in Bone Formation: Highly Efficient Chitosan-pDNA Activated Scaffolds Can Accelerate Bone Regeneration in Critical-Sized Bone Defects

Raftery RM et al. 2017. Biomaterials. 149: 116-127
Application: Bone, Application: Bone/Cartilage

The clinical translation of bioactive scaffolds for the treatment of large segmental bone defects has remained a challenge due to safety and efficacy concerns as well as prohibitive costs. The design of an implantable, biocompatible and resorbable device, which can fill the defect space, allow for cell infiltration, differentiation and neovascularisation, while also recapitulating the natural repair process and inducing cells to lay down new bone tissue, would alleviate the problems with existing treatments. We have developed a gene-activated scaffold platform using a bone-mimicking collagen hydroxyapatite scaffold loaded with chitosan nanoparticles carrying genes encoding osteogenic (BMP-2) and angiogenic (VEGF) proteins. With a single treatment, protein expression by mesenchymal stem cells (MSCs) seeded onto the scaffold is sustained for up to 28 days and is functional in inducing MSC osteogenesis. The in vivo safety and efficacy of this gene-activated scaffold platform was demonstrated resulting in the successful transfection of host cells, abrogating the requirement for multiple procedures to isolate cells or ex vivo cell culture. Furthermore, the level of bone formation at the exceptionally early time-point of 28 days was comparable to that achieved following recombinant BMP-2 protein delivery after 8 weeks in vivo, without the adverse side effects and at a fraction of the cost. This naturally derived cell-free geneactivated scaffold thus represents a new ‘off-the-shelf’ product capable of accelerating bone repair in critical-sized bone defects.