Tissue formation and vascularization in anatomically shaped human joint condyle ectopically in vivo.

Scale-up of bioengineered grafts toward clinical applications is a challenge in regenerative medicine. Here, we report tissue formation and vascularization of anatomically shaped human tibial condyles ectopically with a dimension of 20 x 15 x 15 mm(3). A composite of poly-epsilon-caprolactone and hydroxyapatite was fabricated using layer deposition of three-dimensional interlaid strands with interconnecting microchannels (400 microm) and seeded with human bone marrow stem cells (hMSCs) with or without osteogenic differentiation. An overlaying layer (1 mm deep) of poly(ethylene glycol)-based hydrogel encapsulating hMSCs or hMSC-derived chondrocytes was molded into anatomic shape and anchored into microchannels by gel infusion. After 6 weeks of subcutaneous implantation in athymic rats, hMSCs generated not only significantly more blood vessels, but also significantly larger-diameter vessels than hMSC-derived osteoblasts, although hMSC-derived osteoblasts yielded mineralized tissue in microchannels. Chondrocytes in safranin-O-positive glycosaminoglycan matrix were present in the cartilage layer seeded with hMSC-derived chondrogenic cells, although significantly more cells were present in the cartilage layer seeded with hMSCs than hMSC-derived chondrocytes. Together, MSCs elaborate substantially more angiogenesis, whereas their progenies yield corresponding differentiated tissue phenotypes. Scale up is probable by incorporating a combination of stem cells and their progenies in repeating modules of internal microchannels.

In vitro evaluation of macroporous hydrogels to facilitate stem cell infiltration, growth, and mineralization.

Hydrogels have gained acceptance as biomaterials in a wide range of applications, including pharmaceutical formulations, drug delivery, and tissue sealants. However, exploiting the potential of hydrogels as scaffolds for cell transplantation, tissue engineering, and regenerative medicine still remains a challenge due to, in part, scaffold design limitations. Here, we describe a highly interconnected, macroporous poly(ethylene glycol) diacrylate hydrogel scaffold, with pores ranging from 100 to 600 microm. The scaffold exhibits rapid cell uptake and cell seeding without the need of any external force or device with high incorporation efficiency. When human mesenchymal stem cells are seeded within the porous scaffolds, the scaffolds were found to promote long-term stem cell viability, and on exposure to osteogenic medium, elicit an mineralization response as evaluated by an increased alkaline phosphatase activity (per cell) and calcium and phosphate content within the constructs. The atomic composition of the mineralized matrix was further determined by energy dispersive spectroscopy and found to be similar to calcium-deficient hydroxyapatite, the amorphous biological precursor of bone. The macroporous design of the hydrogel appears advantageous over similar porous hydrogel scaffolds with respect to ease of synthesis, ease of stem cell seeding, and its ability to support long-term stem cell survival and possible differentiation.

Bioengineering strategies to generate vascularized soft tissue grafts with sustained shape.

Tissue engineering offers the possibility for soft tissue reconstruction and augmentation without autologous grafting or conventional synthetic materials. Two critical challenges have been addressed in a number of recent studies: a biology challenge of angiogenesis and an engineering challenge of shape maintenance. These two challenges are inter-related and are effectively addressed by integrated bioengineering strategies. Recently, several integrated bioengineering strategies have been applied to improve bioengineered adipose tissue grafts, including internalized microchannels, delivery of angiogenic growth factors, tailored biomaterials and transplantation of precursor cells with continuing differentiation potential. Bioengineered soft tissue grafts are only clinically meaningful if they are vascularized, maintain shape and dimensions, and remodel with the host. Ongoing studies have begun to demonstrate the feasibility towards an ultimate goal to generate vascularized soft tissue grafts that maintain anatomically desirable shape and dimensions.

Vascularized adipose tissue grafts from human mesenchymal stem cells with bioactive cues and microchannel conduits.

Vascularization is critical to the survival of engineered tissues. This study combined biophysical and bioactive approaches to induce neovascularization in vivo. Further, we tested the effects of engineered vascularization on adipose tissue grafts. Hydrogel cylinders were fabricated from poly(ethylene glycol) diacrylate (PEG) in four configurations: PEG alone, PEG with basic fibroblast growth factor (bFGF), microchanneled PEG, or both bFGF-adsorbed and microchanneled PEG. In vivo implantation revealed no neovascularization in PEG, but substantial angiogenesis in bFGF-adsorbed and/or microchanneled PEG. The infiltrating host tissue consisted of erythrocyte-filled blood vessels lined by endothelial cells, and immunolocalized to vascular endothelial growth factor (VEGF). Human mesenchymal stem cells were differentiated into adipogenic cells, and encapsulated in PEG with both microchanneled and adsorbed bFGF. Upon in vivo implantation subcutaneously in immunodeficient mice, oil red O positive adipose tissue was present and interspersed with interstitial fibrous (IF) capsules. VEGF was immunolocalized in the IF capsules surrounding the engineered adipose tissue. These findings suggest that bioactive cues and/or microchannels promote the genesis of vascularized tissue phenotypes such as the tested adipose tissue grafts. Especially, engineered microchannels may provide a generic approach for modifying existing biomaterials by providing conduits for vascularization and/or diffusion.

Mesenchymal stem cells and tissue engineering.

Mesenchymal stem cells (MSCs) have become one of the most studied stem cells, especially toward the healing of diseased and damaged tissues and organs. MSCs can be readily isolated from a number of adult tissues by means of minimally invasive approaches. MSCs are capable of self-replication to many passages and, therefore, can potentially be expanded to sufficient numbers for tissue and organ regeneration. MSCs are able to differentiate into multiple cell lineages that resemble osteoblasts, chondrocytes, myoblasts, adipocytes, and fibroblasts and express some of the key markers typical of endothelial cells, neuron-like cells, and cardiomyocytes. MSCs have been used alone for cell delivery or seeded in biomaterial scaffolds toward the healing of tissue and organ defects. After an increasing number of the "proof of concept" studies, the remaining tasks are many, such as to determine MSC interactions with host cells and signaling molecules, to investigate the interplay between MSCs and biological scaffold materials, and to apply MSC-based therapies toward clinically relevant defect models. The ultimate goal of MSC-based therapies has valid biological rationale in that clusters of MSCs differentiate to form virtually all connective tissue during development. MSC-based therapies can only be realized our improved understanding of not only their fundamental properties such as population doubling and differentiation pathways but also translational studies that use MSCs in the de novo formation and/or regeneration of diseased or damaged tissues and organs.