The vasculature is an essential component of the circulatory system that plays a vital role in the development, homeostasis, and disease of various organs in the human body. The ability to emulate the architecture and transport function of blood vessels in the integrated context of their associated organs represents an important requirement for studying a wide range of physiological processes. Traditional in vitro models of the vasculature, however, largely fail to offer such capabilities. Here we combine microfluidic three-dimensional (3D) cell culture with the principle of vasculogenic self-assembly to engineer perfusable 3D microvascular beds in vitro. Our system is created in a micropatterned hydrogel construct housed in an elastomeric microdevice that enables coculture of primary human vascular endothelial cells and fibroblasts to achieve de novo formation, anastomosis, and controlled perfusion of 3D vascular networks. An open-top chamber design adopted in this hybrid platform also makes it possible to integrate the microengineered 3D vasculature with other cell types to recapitulate organ-specific cellular heterogeneity and structural organization of vascularized human tissues. Using these capabilities, we developed stem cell-derived microphysiological models of vascularized human adipose tissue and the blood−retinal barrier. Our approach was also leveraged to construct a 3D organotypic model of vascularized human lung adenocarcinoma as a high-content drug screening platform to simulate intravascular delivery, tumor-killing effects, and vascular toxicity of a clinical chemotherapeutic agent. Furthermore, we demonstrated the potential of our platform for applications in nanomedicine by creating microengineered models of vascular inflammation to evaluate a nanoengineered drug delivery system based on active targeting liposomal nanocarriers. These results represent a significant improvement in our ability to model the complexity of native human tissues and may provide a basis for developing predictive preclinical models for biopharmaceutical applications.

Bone substitutes can be designed to replicate physiological structure and function by creating a microenvironment that supports crosstalk between bone and immune cells found in the native tissue, specifically osteoblasts and osteoclasts. Human induced pluripotent stem cells (hiPSC) represent a powerful tool for bone regeneration because they are a source of patient-specific cells that can differentiate into all specialized cell types residing in bone. We show that osteoblasts and osteoclasts can be differentiated from hiPSC-mesenchymal stem cells and macrophages when co-cultured on hydroxyapatite-coated poly(lactic-co-glycolic acid)/poly(L-lactic acid) (HA–PLGA/PLLA) scaffolds. Both cell types seeded on the PLGA/PLLA especially with 5% w/v HA recapitulated the tissue remodeling process of human bone via coupling signals coordinating osteoblast and osteoclast activity and finely tuned expression of inflammatory molecules, resulting in accelerated in vitro bone formation. Following subcutaneous implantation in rodents, co-cultured hiPSC-MSC/-macrophage on such scaffolds showed mature bone-like tissue formation. These findings suggest the importance of coupling matrix remodeling through osteoblastic matrix deposition and osteoclastic tissue resorption and immunomodulation for tissue development.

Tissue adhesives can bind together damaged tissues and serve as tools to deliver and localize therapeutics to facilitate regeneration. One emerging therapeutic trend in orthopedics is the use of intraoperative biologics (IOB), such as bone marrow (BM) and platelet-rich plasma (PRP), to stimulate healing. Here, we introduce the application of the biomaterial chondroitin sulfate succinimidyl succinate (CS-NHS) to deliver IOB in a hydrogel adhesive. We demonstrate the biomaterial's ability to bind various tissue types and its cellular biocompatibility with encapsulated human mesenchymal stem cells (hMSCs). Further, we examine in detail the CS-NHS adhesive combined with BM aspirate for use in bone applications. hMSCs were encapsulated in CS-BM and cultured for 5 weeks in osteogenic medium. Quantitative RT-PCR demonstrated osteogenesis via upregulation of the osteogenic transcription factor Runx2 and bone markers alkaline phosphatase and osteocalcin. Significant deposition of calcium and osteocalcin was detected using biochemical, histological, and immunohistochemical techniques. Shear testing demonstrated that the CS-BM adhesive exhibited an adhesive strength approximately an order of magnitude stronger than fibrin glue and approaching that of a cyanoacrylate adhesive. These results indicate that CS-NHS is a promising delivery tool for IOB in orthopedic applications requiring a strong, degradable, and biocompatible adhesive that supports bone growth. ß

Molecular-genetic and muropeptide analysis techniques have been applied to examine the function in vivo of the Bacillus megaterium QM B1551 SleB and SleL proteins. In common with Bacillus subtilis and Bacillus anthracis, the presence of anhydromuropeptides in B. megaterium germination exudates, which is indicative of lytic transglycosylase activity, is associated with an intact sleB structural gene. B. megaterium sleB cwlJ double mutant strains complemented with engineered SleB variants in which the predicted N-or C-terminal domain has been deleted (SleB-⌬N or SleB-⌬C) efficiently initiate and hydrolyze the cortex, generating anhydromuropeptides in the process. Additionally, sleB cwlJ strains complemented with SleB-⌬N or SleB-⌬C, in which glutamate and aspartate residues have individually been changed to alanine, all retain the ability to hydrolyze the cortex to various degrees during germination, with concomitant release of anhydromuropeptides to the surrounding medium. These data indicate that while the presence of either the N-or C-terminal domain of B. megaterium SleB is sufficient for initiation of cortex hydrolysis and the generation of anhydromuropeptides, the perceived lytic transglycosylase activity may be derived from an enzyme(s), perhaps exclusively or in addition to SleB, which has yet to be identified. B. megaterium SleL appears to be associated with the epimerase-type activity observed previously in B. subtilis, differing from the glucosaminidase function that is apparent in B. cereus/B. anthracis.Spores of the genera Bacillus and Clostridium emerge from dormancy via the process of germination. The germination process comprises a series of sequential biophysical and biochemical reactions that result irreversibly in the spore losing its properties of metabolic dormancy and extreme resistance to various chemical and physical treatments (24, 34). Germination is initiated by the presumed binding of small molecular germinants, commonly amino acids or sugars, to cognate receptors located within the spore inner membrane (25,28). In a process that is poorly understood at the molecular level, this interaction leads to a change in the permeability of the inner membrane, resulting in the release of various solutes from the spore core, including metal ions, calcium dipicolinate (Ca-DPA), and some amino acids (32,33,35). A degree of rehydration of the core is evident at or around the same time, although this is insufficient to permit a significant degree of vegetative metabolism (9, 31). These events, which appear common to all Bacillus species where examined, comprise stage I of germination (31, 32, 34).The major event in stage II of the germination process from a biochemical perspective involves depolymerization of the spore cortex. The spore cortex is a thick layer of peptidoglycan, characterized by the spore-specific muramic acid lactam (MAL) moiety (37, 38), which, together with the thin inner layer of germ cell wall peptidoglycan (36), forms contiguous layers that entirely envelope the spore protoplast. ...