Concomitant control of mechanical properties and degradation in resorbable elastomer-like materials using stereochemistry and stoichiometry for soft tissue engineering

Abstract

Complex biological tissues are highly viscoelastic and dynamic. Efforts to repair or replace cartilage, tendon, muscle, and vasculature using materials that facilitate repair and regeneration have been ongoing for decades. However, materials that possess the mechanical, chemical, and resorption characteristics necessary to recapitulate these tissues have been difficult to mimic using synthetic resorbable biomaterials. Herein, we report a series of resorbable elastomer-like materials that are compositionally identical and possess varying ratios of cis:trans double bonds in the backbone. These features afford concomitant control over the mechanical and surface eroding degradation properties of these materials. We show the materials can be functionalized post-polymerization with bioactive species and enhance cell adhesion. Furthermore, an in vivo rat model demonstrates that degradation and resorption are dependent on succinate stoichiometry in the elastomers and the results show limited inflammation highlighting their potential for use in soft tissue regeneration and drug delivery.

Fig. 1. Stereocontrolled synthesis and characterization of the resorbable elastomers.

A A one-pot thiol-yne step-growth polymerization of propane-1,3-diyl dipropiolate (C_3A, 1) with bis(3-mercaptopropyl) succinate (2) and with 1,6-hexane dithiol (C_6S) forms a copolymer that shows tunable degradation rates depending on the % of amount of repeat unit x (1 + 2) that is incorporated. B The stereochemistry is easily determined by the vinyl proton doublets at δ = 5.7 and 7.7 ppm (trans, 15 Hz) and δ = 5.8 and 7.1 ppm (cis, 9 Hz), respectively. The extent of succinate incorporation will determine both the rate and extent of degradation.

Fig. 2. Compositional-dependent mechanical properties of the resorbable polymers.

A Mass loss as a function bis(3-mercaptopropyl) succinate (2) stoichiometry over time in a series of high cis (78–79%) elastomers show compositionally dependent linear surface erosion behavior. B Increasing the amount of bis(3-mercaptopropyl) succinate (2) which is a longer, bulkier comonomer reduced the UTS of the resulting elastomers. Mechanical properties and degradation rates are highly tunable depending on the amount of cis-alkene bonds in the backbone and stoichiometric control of succinate content. Succinate groups in the chemical structure provide flexibility and hydrophilicity to the polymer chains and facilitate the degradation process. The increase in chain mobility and hydrophilicity results in an increase in the number of degradable ester groups and hence the degradation rates (C) and a decrease of Young’s modulus (D). Increasing the cis-alkene content resulted in slower degradation rates and higher Young’s moduli values, decreased ultimate strain. Error bars represent one standard deviation of the mean (n = 3).

Fig. 3. Surface Erosion and Swelling.

A Discs (4 mm diameter, 0.5 mm thick) were cut from vacuum film compression samples and placed in 1× PBS in the incubator (37 °C, 5% CO₂ humidified atmosphere) for up to 32 days. The data were plotted in three different ways: dry mass change compared to the original mass (degradation), wet mass change compared to the original mass (traditional swelling if no degradation), and wet mass/dry mass at each time point (swelling if degradation). The swelling behavior of the polymers was determined by tracking the wet and dry mass of the disc samples at each time point. Error bars represent one standard deviation of the mean (n = 4). B Analysis of SEM micrographs of the respective test coupons exposed to accelerated degradation conditions indicates uniform degradation and pitting indicative of surface erosion processes. Scale bars = 10 μm.

Fig. 4. Cell Viability and Post polymerization Functionalization.

A Live/Dead® staining of cells incubated on each substrate for 24 h. Calcein-AM was used to stain live cells (green) and ethidium homodimer-1 was used for dead cells (red). Scale Bars are 200 μm. B Representative fluorescence pictures of hMSCs cultured on degradable polymer substrates for 72 h. Scale bars are 500 μm. C Quantitative cell viability data showed >95% viability after 24 h in both hMSCs and MC3T3 fibroblasts. Error bars represent one standard deviation of the mean (n = 5). D Cell metabolic activity showed an increase in approximate cell number on degradable polymer substrates over 7 days. Error bars represent one standard deviation of the mean (n = 5). E To demonstrate the ability to derivatize the polymers post polymerization, a Megastokes®-673-azide dye surrogate specifically binds to the internal alkyne functionalized polymer (F). Scale bar is 500 μm. Cell spreading on (poly(bis(4-(propioloyloxy)but-2-yn-1-yl)-3,3’-(hexane-1,6-diylbis(sulfanediyl)))) films without (G) and with (H) RGD functionalization. Increased cell adhesion, spreading and integrin-associated actin fiber formation was evident in the RGD derivatized films, indicating that RGD conjugation was successful. Scale bars are 100 μm.

Fig. 5. Subcutaneous in vivo degradation of Poly(L-lactic acid) (PLLA), 80% cis/50% succinate and 80% cis/100% succinate over a 4-month timeframe.

Surgical procedures with subcutaneous implantation involved a small incision, polymer disc insertion, and incision closure with Michel-clips. Four samples were implanted per animal (A). (B) Following extraction, the implants can be visualized in the host tissue using Hematoxylin and Eosin (H&E) and Masson’s Trichrome staining. As seen, there are almost no macroscopic indications of an inflammatory response. Whole-mount cross-section images showing thick fibrous encapsulation surrounding PLLA after 4 months of incubation in vivo are observed. Similar behavior to PLLA is observed for 80% cis/50% succinate at 1- and 4-months implantation. Alternatively, the early stages of cellular infiltration are noticed in 80% cis/100% succinate after only 1 month (I). After 2- (Supplementary Fig. 23) and 4- months, noticeable shrinking/resorption of the polymer was seen with continued cellular infiltration. Degradation after 4 months is nearly complete with cells, deposited collagen, and tissue fully encompassing the polymer area. Blood vessel sprouts and multinucleated giant cells are noticeable throughout the polymer space that has been resorbed. Trichome images show collagen deposition and immunohistochemistry staining macrophages for pro-inflammatory (M1), non-activated (M0), and anti-inflammatory (M2) macrophages show degradation induced inflammation and remodeling. Inset scale bar = 200 μm. Shown micrographs are representative of histology specimens (n = 4) from each of six independent implants (n = 6) for each material.

Fig. 6. Picrosirius Red staining of 80cis:100 succinate elastomers after 1 month and 4 months of subcutaneous incubation.

Collagen deposition and maturation occurred throughout the polymer space with different orientations representing both mature and developing collagen through the center of the polymer area. Scale bars are 100 μm. Shown micrographs are representative of histology specimens (n = 4) from each of six independent implants (n = 6) for each material.