Silk-based injectable biomaterial as an alternative to cervical cerclage: an in vitro study

Abstract

Objective: New therapies to prevent preterm birth are needed. Our objective was to study an injectable biomaterial for human cervical tissue as an alternative to cervical cerclage.

Study design: Human cervical tissue specimens were obtained from premenopausal gynecological hysterectomies for benign indications. A 3-part biomaterial was formulated, consisting of silk protein solution blended with a 2-part polyethylene glycol gelation system. The solutions were injected into cervical tissue and the tissue was evaluated for mechanical properties, swelling, cytocompatibility, and histology.

Results: The stiffness of cervical tissue more than doubled after injection (P = .02). Swelling properties of injected tissue were no different than native tissue controls. Cervical fibroblasts remained viable for at least 48 hours when cultured on the biomaterial.

Conclusions: We report a silk-based, biocompatible, injectable biomaterial that increased the stiffness of cervical tissue compared to uninjected controls. Animal studies are needed to assess this biomaterial in vivo.

Keywords: cerclage; cervix; injectable biomaterial; preterm birth; silk.

Figure 1.

Biomaterial formation, gross morphology, and histology. A (left), The 2-step gelation process is shown. Mixing of the 2 solutions results in a chemically cross-linked gel due to the reaction between the thiol and the maleimide functional groups. Further stabilization arises due to β-sheet formation in silk, which occurs when the silk–polyethylene glycol (PEG) is exposed to methanol or ethanol (white color) but not to water or phosphate-buffered saline (PBS). B (upper right), After injection, the cervical tissue appears larger due to the increase in mass. The silk–polyethylene glycol appears as a translucent, white material on the tissue. C (lower right), Hematoxylin and eosin histology of cervical tissue after injection shows integration of the silk–polyethylene glycol biomaterial (purple) into the tissue. For reference to color, please see online version.

Figure 2.

Swelling properties. Swelling properties of tissue injected with polyethylene glycol (PEG) and silk were no different than the native tissue controls. Swelling was significantly increased (P < .01) in the tissue injected with PEG alone.

Figure 3.

Mechanical testing. A (upper left), Setup for indentation testing with the cervical tissue specimen placed in phosphate-buffered saline (PBS). Indentation was performed in the same spot before and after tissue injection. B (upper right), A sample load–unload cycle on a cervical tissue specimen shows a consistent response between the testing cycles (n = 3 cycles). Of note, the sample was preloaded by 4 to 8 mN prior to indentation. C (lower left), The peak force needed to compress the cervical tissue by 20% increased significantly after injection of the silk–polyethylene glycol (PEG) biomaterial. D (lower right), A positive dose response was seen. As more silk–PEG biomaterial was injected, the peak force ratio significantly increased (P = .002).

Figure 4.

Cytocompatibility. Cervical fibroblasts are viable (green cells) when cultured for 48 hours on cervical tissue injected with silk–polyethylene glycol (PEG) biomaterial. Viable cervical fibroblasts display a rounded morphology on the silk–PEG biomaterial and spindle-shaped morphology on cervical tissue (inset). On the silk–PEG biomaterial alone (control), the cervical fibroblasts have a rounded morphology (far right panel). For reference to color, please see online version.