The Balance between the Hemostatic Effect and Immune Response of Hyaluronan Conjugated with Different Chain Lengths of Inorganic Polyphosphate.

Inorganic polyphosphate (PolyP) is a potential hemostatic material. However, the effect of PolyP chain length on the immune response and hemostatic function remains to be established. We have developed PolyP-conjugated hyaluronans (HA-PolyPs) with three different short-chain PolyPs (n = 13, 40, and 100 phosphate units). All short-chain PolyPs showed biocompatibility in the cell viability and inflammatory cytokine secretion test in vitro and in vivo, wherein shorter PolyPs showed milder responses in some cases. We then produced HA-PolyP hydrogels (HAX-PolyPs) with three different short-chain PolyPs as hemostats. Interestingly, the in vivo biocompatibility and hemostatic activity of HAX-PolyP were not significantly affected by the length of conjugated PolyPs. HAX-PolyP with all chain lengths significantly decreased the amount of bleeding in a novel mouse liver bleeding model. These results indicated that the shortest PolyP (n = 13) induced milder acute inflammation and had an efficient hemostatic effect when conjugated to hyaluronic acid. The present study provides key insights into the design of PolyP-based biomaterials and bioconjugates, which are expected to grow in importance for various medical applications.

Injectable Hemostat Composed of a Polyphosphate-Conjugated Hyaluronan Hydrogel.

We have developed a new hydrogel hemostat composed of hyaluronan (HA) conjugated with inorganic polyphosphate (PolyP). A hemostatic hydrogel, HAX-PolyP, was formed rapidly by mixing aldehyde-modified HA and hydrazide-modified HA conjugated with PolyP (HA-PolyP). Although the gelation rate decreased with increasing PolyP content, the gelation time was below 5 min. In addition, the hydrogel swelling volume decreased with increasing PolyP content, but the degradation rate did not depend on PolyP content and the hydrogel underwent complete degradation through hydrolysis over 3 weeks in phosphate buffered saline. HAX-PolyP showed similar biocompatibility with the HA hydrogel without PolyP conjugation in vitro and in vivo. Intraperitoneal administration of HAX-PolyP did not induce any adhesion in the peritoneum and clot formation in the lungs. Finally, HA-PolyP accelerated the coagulation rate of human plasma ex vivo, and HAX-PolyP showed as strong a hemostatic effect as fibrin glue in a mouse liver bleeding model in vivo.

Enhancing osteogenic differentiation of MC3T3-E1 cells by immobilizing inorganic polyphosphate onto hyaluronic acid hydrogel.

In tissue engineering, precise control of cues in the microenvironment is essential to stimulate cells to undergo bioactivities such as proliferation, differentiation, and matrix production. However, current approaches are inefficient in providing nondepleting cues. In this study, we have developed a novel bioactive hydrogel (HAX-PolyP) capable of enhancing tissue growth by conjugating inorganic polyphosphate chains onto hyaluronic acid macromers. The immobilized polyphosphates provided constant osteoconductive stimulation to the embedded murine osteoblast precursor cells, resulting in up-regulation of osteogenic marker genes and enhanced levels of ALP activity. The osteoconductive activity was significantly higher when compared to those stimulated with free-floating polyphosphates. Even at very low concentrations, immobilization of polyphosphates onto the scaffold allowed sufficient signaling leading to more effective osteoconduction. These results demonstrate the potential of our novel material as an injectable bioactive scaffold, which can be clinically useful for developing bone grafts and bone regeneration applications.

Development of carboxymethyl cellulose nonwoven sheet as a novel hemostatic agent.

Carboxymethyl cellulose (CMC) is a plant-derived material that has high biocompatibility and water solubility. We developed a CMC nonwoven sheet as a hemostatic agent by carboxymethylating a continuous filament cellulose nonwoven sheet. The CMC nonwoven sheet was able to absorb water and dissolve in it. The rates of absorption and dissolution depended on the degree of carboxymethylation. After dissolving in blood, CMC accelerated clot development (possibly owing to the incorporation of CMC into fibrin fibers) and increased the viscosity of the blood, both of which would contribute to the improved blood clotting of an injured surface. In vivo experiments using a rat tail cutting method showed that a CMC nonwoven sheet shortened the bleeding time of the tail when applied to the cut surface. The hemostatic effect of the CMC nonwoven sheet was almost at the same level as a commercial hemostatic bandage. These results suggest that a CMC nonwoven sheet could be used as a novel sheet-type hemostatic agent.