Photo-Modulated Therapeutic Protein Release from a Hydrogel Depot Using Visible Light.

The use of biomacromolecular therapeutics has revolutionized disease treatment, but frequent injections are required owing to their short half-life in vivo. Thus there is a need for a drug delivery system that acts as a reservoir and releases the drug remotely "on demand". Here we demonstrate a simple light-triggered local drug delivery system through photo-thermal interactions of polymer-coated gold nanoparticles (AuNPs) inside an agarose hydrogel as therapeutic depot. Localized temperature increase induced by the visible light exposure caused reversible softening of the hydrogel matrix to release the pre-loaded therapeutics. The release profile can be adjusted by AuNPs and agarose concentrations, light intensity and exposure time. Importantly, the biological activity of the released bevacizumab was highly retained. In this study we demonstrate the potential application of this facile AuNPs/hydrogel system for ocular therapeutics delivery through its versatility to release multiple biologics, compatibility to ocular cells and spatiotemporal control using visible light.

Use of Human Lung Tissue Models for Screening of Drugs against SARS-CoV-2 Infection.

The repurposing of licenced drugs for use against COVID-19 is one of the most rapid ways to develop new and alternative therapeutic options to manage the ongoing pandemic. Given circa 7817 licenced compounds available from Compounds Australia that can be screened, this paper demonstrates the utility of commercially available ex vivo/3D airway and alveolar tissue models. These models are a closer representation of in vivo studies than in vitro models, but retain the benefits of rapid in vitro screening for drug efficacy. We demonstrate that several existing drugs appear to show anti-SARS-CoV-2 activity against both SARS-CoV-2 Delta and Omicron Variants of Concern in the airway model. In particular, fluvoxamine, as well as aprepitant, everolimus, and sirolimus, has virus reduction efficacy comparable to the current standard of care (remdesivir, molnupiravir, nirmatrelvir). Whilst these results are encouraging, further testing and efficacy studies are required before clinical use can be considered.

Biocompatibility and immunogenic response to recombinant honeybee silk material.

If tolerated in biological environments, recombinant structural proteins offer the advantage that biological cues dictating cell attachment and material degradation can be modified as required for clinical application using genetic engineering. In this study, we investigate the biological response to materials generated from the recombinant honeybee silk protein, AmelF3, a structural protein that can be produced at high levels by fermentation in Escherichia coli. The protein can be readily purified from E. coli host cell proteins after transgenic production and fabricated into various material formats. When implanted subcutaneously according to International Standard ISO 10993 tests, materials generated from the purified recombinant protein were found to be noncytotoxic, inducing a transient weak immunogenic response and a chronic inflammatory response that resolved over time. While preliminary, this study supports the ongoing development of materials generated from this protein for biomedical applications. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1763-1770, 2019.

Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro.

Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.

Engineering specific chemical modification sites into a collagen-like protein from Streptococcus pyogenes.

Recombinant bacterial collagens provide a new opportunity for safe biomedical materials. They are readily expressed in Escherichia coli in good yield and can be readily purified by simple approaches. However, recombinant proteins are limited in that direct secondary modification during expression is generally not easily achieved. Thus, inclusion of unusual amino acids, cyclic peptides, sugars, lipids, and other complex functions generally needs to be achieved chemically after synthesis and extraction. In the present study, we have illustrated that bacterial collagens that have had their sequences modified to include cysteine residue(s), which are not normally present in bacterial collagen-like sequences, enable a range of specific chemical modification reactions to be produced. Various model reactions were shown to be effective for modifying the collagens. The ability to include alkyne (or azide) functions allows the extensive range of substitutions that are available via "click" chemistry to be accessed. When bifunctional reagents were used, some crosslinking occurred to give higher molecular weight polymeric proteins, but gels were not formed. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 806-813, 2017.

Ovine multiparity is associated with diminished vaginal muscularis, increased elastic fibres and vaginal wall weakness: implication for pelvic organ prolapse.

Pelvic Organ Prolapse (POP) is a major clinical burden affecting 25% of women, with vaginal delivery a major contributing factor. We hypothesised that increasing parity weakens the vagina by altering the extracellular matrix proteins and smooth muscle thereby leading to POP vulnerability. We used a modified POP-quantification (POP-Q) system and a novel pressure sensor to measure vaginal wall weakness in nulliparous, primiparous and multiparous ewes. These measurements were correlated with histological, biochemical and biomechanical properties of the ovine vagina. Primiparous and multiparous ewes had greater displacement of vaginal tissue compared to nulliparous at points Aa, Ap and Ba and lower pressure sensor measurements at points equivalent to Ap and Ba. Vaginal wall muscularis of multiparous ewes was thinner than nulliparous and had greater elastic fibre content. Collagen content was lower in primiparous than nulliparous ewes, but collagen organisation did not differ. Biomechanically, multiparous vaginal tissue was weaker and less stiff than nulliparous. Parity had a significant impact on the structure and function of the ovine vaginal wall, as the multiparous vaginal wall was weaker and had a thinner muscularis than nulliparous ewes. This correlated with "POP-Q" and pressure sensor measurements showing greater tissue laxity in multiparous compared to nulliparous ewes.

Adipose tissue engineering based on the controlled release of fibroblast growth factor-2 in a collagen matrix.

Adipose tissue forms when basement membrane extract (Matrigel) and fibroblast growth factor-2 (FGF-2) are added to our mouse tissue engineering chamber model. A mouse tumor extract, Matrigel is unsuitable for human clinical application, and finding an alternative to Matrigel is essential. In this study we generated adipose tissue in the chamber model without using Matrigel by controlled release of FGF-2 in a type I collagen matrix. FGF-2 was impregnated into biodegradable gelatin microspheres for its slow release. The chambers were filled with these microspheres suspended in 60 microL collagen gel. Injection of collagen containing free FGF-2 or collagen containing gelatin microspheres with buffer alone served as controls. When chambers were harvested 6 weeks after implantation, the volume and weight of the tissue obtained were higher in the group that received collagen and FGF-2 impregnated microspheres than in controls. Histologic analysis of tissue constructs showed the formation of de novo adipose tissue accompanied by angiogenesis. In contrast, control groups did not show extensive adipose tissue formation. In conclusion, this study has shown that de novo formation of adipose tissue can be achieved through controlled release of FGF-2 in collagen type I in the absence of Matrigel.

Real-time measurement of the vaginal pressure profile using an optical-fiber-based instrumented speculum.

Pelvic organ prolapse (POP) occurs when changes to the pelvic organ support structures cause descent or herniation of the pelvic organs into the vagina. Clinical evaluation of POP is a series of manual measurements known as the pelvic organ prolapse quantification (POP-Q) score. However, it fails to identify the mechanism causing POP and relies on the skills of the practitioner. We report on a modified vaginal speculum incorporating a double-helix fiber-Bragg grating structure for distributed pressure measurements along the length of the vagina and include preliminary data in an ovine model of prolapse. Vaginal pressure profiles were recorded at 10 Hz as the speculum was dilated incrementally up to 20 mm. At 10-mm dilation, nulliparous sheep showed higher mean pressures ( 102 ± 46 ?? mmHg ) than parous sheep ( 39 ± 23 ?? mmHg ) ( P = 0.02 ), attributable largely to the proximal (cervical) end of the vagina. In addition to overall pressure variations, we observed a difference in the distribution of pressure that related to POP-Q measurements adapted for the ovine anatomy, showing increased tissue laxity in the upper anterior vagina for parous ewes. We demonstrate the utility of the fiber-optic instrumented speculum for rapid distributed measurement of vaginal support.

Formation of multimers of bacterial collagens through introduction of specific sites for oxidative crosslinking.

A range of non-animal collagens has been described, derived from bacterial species, which form stable triple-helical structures without the need for secondary modification to include hydroxyproline in the sequence. The non-animal collagens studied to date are typically smaller than animal interstitial collagens, around one quarter the length and do not pack into large fibrillar aggregates like those that are formed by the major animal interstitial collagens. A consequence of this for biomedical products is that fabricated items, such as collagen sponges, are not as mechanically and dimensionally stable as those of animal collagens. In the present study, we examined the production of larger, polymeric forms of non-animal collagens through introduction of tyrosine and cysteine residues that can form selective crosslinks through oxidation. These modifications allow the formation of larger aggregates of the non-animal collagens. When Tyr residues were incorporated, gels were obtained. And with Cys soluble aggregates were formed. These materials can be formed into sponges that are more stable than those formed without these modifications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2369-2376, 2016.

Evaluation of an established pericardium patch for delivery of mesenchymal stem cells to cardiac tissue.

The present study has evaluated a commercial pericardial material for its capacity to assist as a natural extracellular matrix (ECM) patch for the delivery and retention of mesenchymal stem cells for cardiac repair. The repair of cardiac tissue with cells delivered by an appropriate bioscaffold is expected to offer a superior, long-lasting treatment strategy. The present material, CardioCel®, is based on acellular pericardium that has been stabilized by treatments, including a low concentration of glutaraldehyde, that eliminate calcification after implantation. In the present study, we have assessed this material using human bone marrow mesenchymal stem cells at various cell densities under standard, static cell culture conditions. The initial seeding densities were monitored to evaluate the extent of cell attachment and cell viability, with subsequent cell proliferation assessed up to 4 weeks using an MTS assay. Cell morphology, infiltration, and spreading were tracked using scanning electron microscopy and phalloidin staining. The efficacy of long-term cell survival was further assessed by examining the extent and type of new tissue formation on seeded scaffolds at 70 days; both type I and type III collagens were present in fibrillar structures on these scaffolds indicating that the seeded stem cells had the capacity to differentiate into collagen-producing cells necessary to repair damaged ECM. These data show that the CardioCel® scaffold is an appropriate substrate for the stem cells and has the potential to both retain seeded stem cells and to act as a template for cell propagation and new tissue formation.

Controlled surface modification of tissue culture polystyrene for selective cell binding using resilin-inspired polypeptides.

Modified tissue culture polystyrene (TCP) surfaces have been fabricated by attachment of recombinant polypeptides based on Drosophila melanogaster resilin and the Anopheles gambiae resilin-like protein. The D. melanogaster polypeptide (Rec-1) was from the first exon of resilin and consisted of 17 very similar repeats of a 15 residue sequence. The A. gambiae polypeptide consisted of 16 repeats of an 11 residue consensus sequence (An16). Polypeptides were attached to the TCP surface through tyrosine-based photo-crosslinking using blue light in combination with (RuII(bpy)3)Cl2 and sodium persulfate. TCP that has been manufactured by mild oxidation has surface phenolic groups that are believed to participate in this crosslinking process. X-ray photoelectron spectroscopy and contact angle analyses were used to demonstrate polypeptide binding. At higher coating concentrations of Rec-1 and An16, the surface was passivated and fibroblasts no longer attached and spread. At coating concentrations of 1 mg ml(-1) for Rec-1 and 0.1 mg ml(-1) for An16, where the surface was fully passivated against fibroblast attachment, addition of a cell attachment peptide, cyclo(Arg-Gly-Asp-D-Tyr-Lys) during coating and photo-crosslinking at >0.1 mg ml(-1), led to the restoration of fibroblast binding that was dependent on the integrin αV chain.

Glycomic analyses of ovarian follicles during development and atresia.

To examine the detailed composition of glycosaminoglycans during bovine ovarian follicular development and atresia, the specialized stromal theca layers were separated from the stratified epithelial granulosa cells of healthy (n=6) and atretic (n=6) follicles in each of three size ranges: small (3-5mm), medium (6-9mm) and large (10mm or more) (n=29 animals). Fluorophore-assisted carbohydrate electrophoresis analyses (on a per cell basis) and immunohistochemistry (n=14) were undertaken. We identified the major disaccharides in thecal layers and the membrana granulosa as chondroitin sulfate-derived ∆uronic acid with 4-sulfated N-acetylgalactosamine and ∆uronic acid with 6-sulfated N-acetylgalactosamine and the heparan sulfate-derived Δuronic acid with N-acetlyglucosamine, with elevated levels in the thecal layers. Increasing follicle size and atresia was associated with increased levels of some disaccharides. We concluded that versican contains 4-sulfated N-acetylgalactosamine and it is the predominant 4-sulfated N-acetylgalactosamine proteoglycan in antral follicles. At least one other non- or 6-sulfated N-acetylgalactosamine proteoglycan(s), which is not decorin or an inter-α-trypsin inhibitor family member, is present in bovine antral follicles and associated with hitherto unknown groups of cells around some larger blood vessels. These areas stained positively for chondroitin/dermatan sulfate epitopes [antibodies 7D4, 3C5, and 4C3], similar to stem cell niches observed in other tissues. The sulfation pattern of heparan sulfate glycosaminoglycans appears uniform across follicles of different sizes and in healthy and atretic follicles. The heparan sulfate products detected in the follicles are likely to be associated with perlecan, collagen XVIII or betaglycan.

Stabilization of collagen tissues by photocrosslinking.

Photocrosslinking, using 2 mM Ru(II)(bpy)(3)Cl(2) and various concentrations of sodium persulfate with irradiation by blue light, ∼455 nm, has been shown to be a rapid and effective method for crosslinking various tissues: tendon, amnion membrane, pericardium, and heart valve leaflet. The presence of new crosslinking was demonstrated by the increase in the shrinkage temperature of these tissues. In all the cases, increase in the shrinkage temperatures were seen, although at higher sodium persulfate concentrations, for example, 100 mM, both with and without the Ru(II)(bpy)(3)Cl(2) catalyst, some degradation of the collagenous tissues was found. The effectiveness of this photocrosslinking method when used with tissues was also shown through the increase in the break strength of tissues after crosslinking, and by the reduction of protein that could be extracted by urea. In solution studies, dityrosine has been shown to be formed during photocrosslinking. With tissues, Western blotting showed the presence of new dityrosine crosslinked proteins.

Effect of local, long-term delivery of platelet-derived growth factor (PDGF) on injected fat graft survival in severe combined immunodeficient (SCID) mice.

SUMMARY BACKGROUND: Autogenous fat injection is widely used for the correction of acquired and congenital soft tissue defects. However, the high absorption rate results in the need for over-correction of the defect and repeat procedures. We hypothesised that platelet-derived growth factor (PDGF), a potent mitogen and known stimulant for murine preadipocytes, would improve fat graft survival when concentrations were sustained with a gelatine microsphere delivery system. METHODS: Abdominal fat was harvested from an otherwise healthy 43-year-old woman during a breast reconstruction. Prior to subdermal injection into severe combined immunodeficient (SCID) mice, the fat grafts were divided into 1-ml aliquots, mixed with microspheres bound to PDGF, free PDGF, or nothing depending on its experimental group, and weighed. The following experimental groups were thus created (minimum n=8 per group): (1) fat graft control, (2) fat graft with free PDGF, (3) fat graft with blank microspheres, and (4) fat graft with microspheres bound to PDGF. After 12 weeks, the fat xenografts were harvested for analysis of weight maintenance and histological and morphometric evaluation. RESULTS: The addition of PDGF bound to gelatine microspheres was effective in improving xenograft weight maintenance (P=0.018) and preservation of adipose tissue architecture (P<0.0005) compared to controls at 3 months. The microspheres were completely absorbed at 12 weeks. CONCLUSIONS: Sustained, local delivery of PDGF via a gelatine microsphere delivery system resulted in improved weight maintenance of the xenografts with greater preservation of adipose tissue architecture at 3 months compared to controls.