(H-291) Calcium-scavenging and anti-inflammatory hydrogels to prevent pathologic bone formation

Heterotopic ossification (HO), or the formation of pathologic bone tissue outside the skeleton, is a debilitating condition that affects both Service Members and civilians following traumatic orthopedic injuries. These bony nodules often arise following blast injuries or at sites of limb amputation and can cause significant pain, joint immobilization, or soft tissue damage. Since HO does not regress over time, patients with advanced heterotopic bone formation typically require extensive surgeries to relieve symptoms. Though some preventative treatments for HO such as radiotherapy or bisphosphonate drug treatment have shown promise, these interventions are inconsistent and often hinder normal tissue healing. In short, there are no wholly effective treatments for inhibiting HO development in current clinical practice. Based on these limitations with current HO prevention strategies, this work describes the development injectable hydrogels that can be prophylactically administered directly to an injury site to both reprogram the local inflammatory tissue environment and remove calcium ion precursors that form heterotopic bone. Crucially, these minimally-invasive biomaterials will mitigate multiple HO-linked factors while avoiding systemic side effects.

Poly(aspartic acid) (PASA) is a synthetic polypeptide that is highly hydrophilic, biocompatible, easily degraded by proteases, and features a strong affinity for calcium ions. In biomedical applications, PASA’s attraction to calcium has often been employed to create drug delivery vehicles that can target calcified bone tissue but has never been explored to prevent biological calcium deposition within the body. Here, injectable PASA hydrogel therapies are developed with the goal of preventing localized tissue calcifications which drive HO formation. The precursor to linear PASA is poly(succinimide) (PSI), which is generated by a thermal condensation of L-aspartic acid and an acid catalyst. PSI reacts with primary amine groups and was functionalized with cysteamine (H2N-CH2CH2-SH) to generate side chains with free thiol groups. Next the thiolated PSI is ring-opened under basic conditions to generate thiol-functionalized PASA which can be added with a poly(ethylene glycol) (PEG) 4-arm macromer featuring thiol-reactive maleimide end groups (PEG-MAL) to generate fast-gelling hydrogels. These hydrogels were first incubated in either saline or a collagenase solution (3mg/mL) over 10 days and weighed for both wet and dry mass to confirm biodegradability. Next, the antioxidant compound 4-amine TEMPO was covalently conjugated onto the PASA precursor polymer PSI and assessed for radical scavenging against naïve PSI using a a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Finally, PASA hydrogels or non-PASA gel controls were incubated with calcium chloride solutions to assess calcium ion scavenging using titration with Eriochrome Black T / ethylenediamine tetraacetic acid (EDTA).

As expected from previous literature with hydrogels formed with PEG-MAL macromers, the PASA hydrogels formed in less than 30 sec when mixing the two components as shown in Figure 1A. This fast gelation makes these materials amenable for minimally-invasive injection into patients who are at serious risk for developing heterotopic bone formation. Next, the biodegradability of these materials was confirmed after incubating gels in either saline of the natural enzyme collagenase. As expected, the gels were relatively unchanged in saline but underwent rapid degradation when incubated with the enzyme as shown in Figure 1B. The tunability of PASA polymers based on conjugating amine-containing molecules was also utilized for the addition of the well-known antioxidant compound 4-amino TEMPO. The amine-containing TEMPO molecule was added to PASA precursor PSI and assessed for inhibition of the radical molecule DPPH, demonstrating potent antioxidant activity compared to unmodified PSI (Figure 1C). Finally and most crucially, the calcium scavenging ability of the PASA hydrogels was determined by incubating PASA and non-PASA control gels with free calcium ions. Though passive diffusion allowed some calcium to be retained in the non-PASA controls as shown in Figure 1D, there was a significant PASA dose-dependent increase in calcium scavenging in the bioactive hydrogel formulations. These preliminary data confirm our fundamental hypothesis of PASA hydrogel calcium scavenging and motivate the further development of this platform. This technology represents an innovative departure from HO prevention strategies seen in either the clinic or in experimental preclinical investigations. This newly proposed hydrogel platform is a potentially paradigm-shifting intervention that uses minimally invasive delivery of a calcium-scavenging biomaterial to prevent heterotopic bone formation at specific orthopaedic injury sites.