Potential of Thermolysin-like Protease A69 in Preparation of Bovine Collagen Peptides with Moisture-Retention Ability and Antioxidative Activity

Bovine bone is rich in collagen and is a good material for collagen peptide preparation. Although thermolysin-like proteases (TLPs) have been applied in different fields, the potential of TLPs in preparing bioactive collagen peptides has rarely been evaluated. Here, we characterized a thermophilic TLP, A69, from a hydrothermal bacterium Anoxybacillus caldiproteolyticus 1A02591, and evaluated its potential in preparing bioactive collagen peptides. A69 showed the highest activity at 60 °C and pH 7.0. We optimized the conditions for bovine bone collagen hydrolysis and set up a process with high hydrolysis efficiency (99.4%) to prepare bovine bone collagen peptides, in which bovine bone collagen was hydrolyzed at 60 °C for 2 h with an enzyme–substrate ratio of 25 U/g. The hydrolysate contained 96.5% peptides that have a broad molecular weight distribution below 10000 Da. The hydrolysate showed good moisture-retention ability and a high hydroxyl radical (•OH) scavenging ratio of 73.2%, suggesting that the prepared collagen peptides have good antioxidative activity. Altogether, these results indicate that the thermophilic TLP A69 has promising potential in the preparation of bioactive collagen peptides, which may have potentials in cosmetics, food and pharmaceutical industries. This study lays a foundation for the high-valued utilization of bovine bone collagen.

Delayed chemical meningitis after Chiari decompression with duraplasty: illustrative cases

BACKGROUND Dural reconstruction to achieve expansion duraplasty is important in suboccipital decompression for Chiari malformation type 1 (CM1). Although various dural substitutes are available, including synthetic collagen matrix grafts and dural xenografts, they have the potential to induce an inflammatory response. In this case series, the authors present their experience and discuss the incidence and possible mechanism of aseptic meningitis after the use of bovine collagen matrix graft as a dural substitute in patients with CM1 after suboccipital decompression. OBSERVATIONS Three consecutive adult female patients who underwent suboccipital decompression at a single institution by a single neurosurgeon were retrospectively reviewed. They all presented with signs of aseptic meningitis in a delayed fashion, responded well to steroid administration, but had recurrence of their symptoms. Bovine collagen dural substitutes are resorbed in a process that induces an inflammatory response manifesting with signs of aseptic meningitis and is only alleviated with removal of the dural substitute. LESSONS DuraMatrix Suturable, a dural xenograft derived from bovine dermis, though a viable choice for dural repair, is a potential cause of chemical meningitis after duraplasty in Chiari decompression surgery. In patients presenting with delayed and persistent aseptic meningitis after intervention, removal of this dural substitute led to improved symptomatology.

New Methods of Reconstruction for Old Challenges: The Use of the Integra Graft in Necrotizing Soft Tissue Infections of the Male Genitalia

In this paper, we describe two cases of Fournier’s gangrene (FG) in which Integra grafting was used for reconstruction. FG is a progressive necrotizing infection occurring in the perineal region and on the external genitalia. Reconstructive options using local tissue are limited due to the destruction this infection imposes on the soft tissue. Integra graft is a bilaminate artificial dermis made of shark chondroitin 6-sulfate and bovine collagen. It is applied to the wound bed after debridement and establishment of a healthy, well-vascularized wound base. The patients in this case series had large defects which could not be closed primarily with tissue beds and would not have been appropriate for skin grafting. Therefore, an Integra graft was placed. In both patients, the wound beds were appropriate for skin grafting after three weeks. Without the Integra graft, both of our patients would have needed to wait a considerable amount of time prior to reconstruction. Our case series further illustrates and supports the use of Integra grafts in such a scenario following Fournier’s gangrene which has only previously been published on three occasions, all of which demonstrated successful outcomes.

Thermosensitive collagen/fibrinogen gels loaded with decorin suppress lesion site cavitation and promote functional recovery after spinal cord injury

The treatment of spinal cord injury (SCI) is a complex challenge in regenerative medicine, complicated by the low intrinsic capacity of CNS neurons to regenerate their axons and the heterogeneity in size, shape and extent of human injuries. For example, some contusion injuries do not compromise the dura mater and in such cases implantation of preformed scaffolds or drug delivery systems may cause further damage. Injectable in situ thermosensitive scaffolds are therefore a less invasive alternative. In this study, we report the development of a novel, flowable, thermosensitive, injectable drug delivery system comprising bovine collagen (BC) and fibrinogen (FB) that forms a solid BC/FB gel (Gel) immediately upon exposure to physiological conditions and can be used to deliver reparative drugs, such as the naturally occurring anti-inflammatory, anti-scarring agent Decorin, into adult rat spinal cord lesion sites. In dorsal column lesions of adult rats treated with the Gel + Decorin, cavitation was completely suppressed and instead lesion sites became filled with injury-responsive cells and extracellular matrix materials, including collagen and laminin. Decorin increased the intrinsic potential of dorsal root ganglion neurons (DRGN) by increasing their expression of regeneration associated genes (RAGs), enhanced local axon regeneration/sprouting, as evidenced both histologically and by improved electrophysiological, locomotor and sensory function recovery. These results suggest that this drug formulated, injectable hydrogel has the potential to be further studied and translated into the clinic.

Changes in the Molecular Characteristics of Bovine and Marine Collagen in the Presence of Proteolytic Enzymes as a Stage Used in Scaffold Formation

Biopolymers, in particular collagen and fibrinogen, are the leading materials for use in tissue engineering. When developing technology for scaffold formation, it is important to understand the properties of the source materials as well as the mechanisms that determine the formation of the scaffold structures. Both factors influence the properties of scaffolds to a great extent. Our present work aimed to identify the features of the molecular characteristics of collagens of different species origin and the changes they undergo during the enzymatic hydrolysis used for the process of scaffold formation. For this study, we used the methods of gel-penetrating chromatography, dynamic light scattering, reading IR spectra, and scanning electron microscopy. It was found that cod collagen (CC) and bovine collagen (BC) have different initial molecular weight parameters, and that, during hydrolysis, the majority of either type of protein is hydrolyzed by the proteolytic enzymes within the first minute. The differently sourced collagen samples were also hydrolyzed with the formation of two low molecular fractions: Mw ~ 10 kDa and ~20 kDa. In the case of CC, the microstructure of the final scaffolds contained denser, closely spaced fibrillar areas, while the BC-sourced scaffolds had narrow, short fibrils composed of unbound fibers of hydrolyzed collagen in their structure.

A fructosylated peptide derived from a collagen II T cell epitope for long-term treatment of arthritis (FIA-CIA) in mice

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease which affects primarily the joints. Peptides of several proteins have shown an effect in some experimental animal models of RA. We investigated arthritis development in male DBA/1 mice which were injected with bovine collagen II (bCII) and human fibrinogen (hFib) on days 0 and 21, leading to stable and reproducible disease induction in 100% of immunized mice (FIA-CIA). In a second study, two bCII—derived peptides were given three times in the course of 6 weeks after FIA-CIA induction to test for impact on arthritis. Mice were scored weekly for arthritis and anti-citrullinated peptide antibodies (ACPAs) were determined in the sera taken on days 0, 14, 35, 56 and 84. Histology of the hind paws was performed at the end of the experiment. Intravenous administration of peptide 90578, a novel fructosylated peptide derived from the immunodominant T cell epitope of bCII, at a dosage of 1 mg/kg resulted in significant beneficial effects on clinical outcome parameters and on the arthritis histology scores which was sustained over 12 weeks. Survival tended to be improved in peptide 90578-treated mice. Intravenous administration of pure soluble peptide 90578 without adjuvants is a promising approach to treat RA, with treatment starting at a time when ACPAs are already present. The results complement existing data on peptide “vaccination” of healthy animals, or on treatment using recombinant peptide expressing virus or complex biological compounds.

Rapid spheroid assays in a 3-dimensional cell culture chip

Objective The spheroid model provides a physiological platform to study cancer cell biology and drug sensitivity. Usage of bovine collagen I for spheroid assays is costly especially when experiments are conducted in 24-well plates, as high volume of bovine collagen I is needed. The aim of the study was to downsize spheroid assays to a microfluidic 3D cell culture chip and compare the growth, invasion and response to drug/compound of spheroids embedded in the 3D chip to spheroids embedded in 24-well plates. Results Spheroids generated from nasopharyngeal carcinoma cell line HK-1 continuously grew and invaded into collagen matrix in a 24-well plate. Similar observations were noticed with spheroids embedded in the 3D chip. Large spheroids in both 24-well plate and the 3D chip disintegrated and invaded into the collagen matrix. Preliminary drug sensitivity assays showed that the growth and invasion of spheroids were inhibited when spheroids were treated with combination of cisplatin and paynantheine at high concentrations, in a 24-well plate. Comparable findings were obtained when spheroids were treated with the same drug combination in the 3D chip. Moving forward, spheroid assays could be performed in the 3D chip in a more high-throughput manner with minimal time and cost.