Adv Funct Mater. 2016 Aug 23;26(32):5873-5883. doi: 10.1002/adfm.201601146 

Multi-Material Tissue Engineering Scaffolds with Hierarchical Pore Architecture.

Morgan, KY1, Sklaviadis, D1, Tochka, ZL1, Fischer, KM1, Morgan, TD2, Hearon, K1, Langer, R1, and Freed, LE1,3.

  1. Institute for Medical Engineering & Science and the David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
  2. Harvard University School of Engineering & Applied Science, Cambridge, MA 02138, USA
  3. Materials Engineering Division, Draper, Cambridge, MA 02139, USA

Please address correspondence to Lisa E. Freed, 77 Mass. Ave., 76-653, Cambridge, MA 02139, E-mail: lfreed@mit.edu

 

Abstract:

Multi-material polymer scaffolds with multiscale pore architectures were characterized and tested with vascular and heart cells as part of a platform for replacing damaged heart muscle. Vascular and muscle scaffolds were constructed from a new material, poly(limonene thioether) (PLT32i), which met the design criteria of slow biodegradability, elastomeric mechanical properties, and facile processing. The vascular-parenchymal interface was a poly(glycerol sebacate) (PGS) porous membrane that met different criteria of rapid biodegradability, high oxygen permeance, and high porosity. A hierarchical architecture of primary (macroscale) and secondary (microscale) pores was created by casting the PLT32i prepolymer onto sintered spheres of poly(methyl methacrylate) (PMMA) within precisely patterned molds followed by photocuring, de-molding, and leaching out the PMMA. Pre-fabricated polymer templates were cellularized, assembled, and perfused in order to engineer spatially organized, contractile heart tissue. Structural and functional analyses showed that the primary pores guided heart cell alignment and enabled robust perfusion while the secondary pores increased heart cell retention and reduced polymer volume fraction.

 

 

Supplement:

Cardiovascular disease remains the leading cause of death in developed countries (1). In addition, approximately one percent of newborns and an increasing number of adults live with congenital heart disease (2). Pharmacological therapies modulate but cannot replace dysfunctional cardiac tissue, whereas experimental cell therapies and tissue engineered grafts have been shown to improve outcomes (3, 4). An implantable biodegradable elastomer, poly(glycerol-co-sebacate) (PGS), was invented Wang et al. (5, 6) based on the reasoning that implants constructed of non-permanent, compliant materials could provide long term function and integration in a mechanically dynamic in vivo environment. Thereafter, elastomeric polymer scaffolds rendered in PGS and other PGS-inspired materials have been applied to cardiac tissue engineering research (7-10). However, clinical translation of polymer-based grafts for myocardial repair remains limited due to insufficient engineered tissue organization, integration, and scalability. Toward an ultimate goal of creating myocardial grafts for treating injured heart muscle, our work has focused on the design, fabrication, and testing of novel polymer scaffolds capable of directing the functional assembly of cultured heart and vascular cells, as schematically shown in Figure 1.

 

 

Figure 1. Schematic diagram of a polymer platform that supports microvessel perfusion and contractile heart muscle formation in vitro and, if validated in vivo, may aid in the regenerative repair of damaged heart muscle. (A) flow layer, (B) flow layer seeded with vascular cells (red), (C) muscle template added to flow layer and seeded with heart cells (green), (D) flow layer, perfused during cell culture, (E) tissue engineered construct, implanted as a myocardial patch.

 

In early studies, heart cells cultured on PGS grids with rectangular through-pores elicited cell behaviors (elongation and interconnectivity) that appeared to be guided by the scaffold pore pattern (8, 11). Reasoning that multi-layered scaffolds with 3D structural order could direct the behavior of cultured heart cells, we assembled the polymer grids into multi-layered structures with precisely controlled 3D pore architectures (12). In vitro studies of cultured myoblasts and heart cells on the multi-layered PGS scaffolds confirmed the hypothesis that scaffold 3D structural order could differentially direct the elongation of cultured heart cells with respect to the long axes of the rectangular pores (12). Also, tensile mechanical testing demonstrated that scaffold mechanical anisotropy was governed by the rectangular pore shape of the PGS grids.

Related studies explored whether scaffolds with multiple compartments — parenchymal and vascular — could provide a suitable framework to support the formation of engineered tissues (13, 14). Our microfluidic device consisted of a square central area comprising 150 parallel microchannels at the center of a boat-shaped footprint with transitional entrance and exit regions that aided in the distribution of flow. The device was fabricated by using an etched silicon wafer to micromold a polymer base part which was then bonded to a thin PGS covering membrane. In vitro studies were carried out using perfused microfluidic devices with human skeletal muscle cells cultured in parenchymal compartments constructed on top of the covering membrane (13). When the perfusate was standard culture medium the cells in the parenchymal compartment fused into viable myotubes, however if the perfusate was supplemented with a myotoxic drug (doxorubicin) a 90% reduction in myocyte viability was readily induced clearly demonstrating vascular-parenchymal transport through the PGS interface.

The current study, by Morgan et al. (15), built on the aforementioned findings and additionally explored multi-material polymer scaffolds with hierarchical pore structures. Because previous studies had shown that PGS templates degraded too quickly to provide long term structural support to damaged myocardium (16, 17), we combined PGS with a newly invented more slowly degrading elastomeric polymer: poly(limonene thioether) (PLT32i). Microscale pores, introduced using sintered poly(methyl methacrylate) microspheres, were introduced into PGS and PLT32i in an effort to reduce polymer volume fraction and increase oxygen transport. Micropore templating of PGS and PLT32i membranes dramatically increased oxygen permeance as compared with solid polymer membranes, and microporous PGS exhibited the highest oxygen permeance. Based on these degradation and transport data, microporous PGS was selected as a rapidly biodegrading, oxygen permeable vascular-parenchymal interface, and PLT32i fabricated with both macro- and micro-scale pores was selected as a slowly biodegrading template to guide the organized formation of heart muscle and microvessels.

In vitro studies of co-cultured heart and endothelial cells on multi-material (PLT32i-PGS) scaffolds with hierarchical pore structures, performed as shown in Figure 1 A-D, demonstrated cell retention and functionality over five days of perfusion culture (15). Endothelial cells were seen lining the flow channels and utilizing most of the available surface area. Heart cells were found to be aligned predominately in parallel to the long axis of the rectangular pores, consistent with our earlier finding for heart cells on PGS grids (12), and exhibited functional connectivity and synchronous contractility as demonstrated using confocal microscopy and optical mapping. To the best of our knowledge, this is the first report of scaffolds built from two surface-eroding elastomers: PLT32i, newly designed to be photocurable and slowly biodegrading, and PGS, known to be biocompatible and rapidly biodegrading. Moreover, the scaffolds were successfully cellularized, assembled, and perfused to create contractile engineered cardiac tissue. The PLT32i provided hierarchical pore architectures that enabled perfusion and endothelialization while also guiding heart cell alignment and supporting heart cell function. The PGS served as a vascular-parenchymal interface that provided high oxygen permeance.

A pilot study (unpublished) was done in immunodeficient (nude) rats in which two microporous PLT32i grids and a microporous PGS interface were assembled and implanted with or without exogenous heart cells in the setting of a myocardial infarction. Briefly, the animal was anesthetized, intubated, ventilated and maintained on anesthesia. Following thoracotomy and ligation of the left anterior descending coronary artery an implant was loosely sutured to the infarcted region of the left ventricle as shown schematically in Figure 1E. Four weeks after implantation, macroscopic inspection of hearts confirmed the implants had remained in place on the myocardium. Histological analysis revealed host cell infiltration of the pores of the PLT32i scaffolds although the exogenous cardiomyocytes could not be detected. The PLT32i scaffolds exhibited some surface erosion but maintained their structural integrity whereas the PGS porous interfaces had completely biodegraded. This pilot study a first step in exploring the feasibility of using the envisioned scaffold platform in a rodent model of myocardial repair.

Together, the findings suggest that biodegradable elastomeric polymer templates with 3D structural order, hierarchical pore architecture, and mechanical anisotropy can provide a new paradigm for treating injured myocardium by enabling (i) mechanical support and (ii) polymer template-directed engineering of contractile tissue comprising organized heart and vascular cells. With further validation in animal and clinical trials, polymer-based platforms may aid the regenerative repair of damaged heart muscle.

 

Acknowledgment: This work was funded by NIH/NHLBI Grant R01-HL107503.

 

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