Switch of macrophage fusion competency by 3D matrices.

Foreign body reaction reflects the integration between biomaterials and host cells. At the implantation microenvironment, macrophages usually fuse into multinuclear cells, also known as foreign body giant cells, to respond to the biomaterial implants. To understand the biomaterial-induced macrophage fusion, we examined whether biomaterial alone can initiate and control the fusion rate without exogenous cytokines and chemicals. We introduced a collagen-based 3D matrix to embed Raw264.7 cell line and primary rat bone marrow-derived macrophages. We found the biomaterial-stimuli interacted regional macrophages and altered the overall fusogenic protein expressions to regulate the macrophage fusion rate. The fusion rate could be altered by modulating the cell-matrix and cell-cell adhesions. The fused macrophage morphologies, the nuclei number in the fused macrophage, and the fusion rates were matrix dependent. The phenomena were also observed in the in vivo models. These results suggest that the biomaterial-derived stimuli exert similar functions as cytokines to alter the competency of macrophage fusion as well as their drug sensitivity in the biomaterial implanted tissue environment. Furthermore, this in vitro 3D-matrix model has the potential to serve as a toolbox to predict the host tissue response on implanted biomaterials.

PEGylated Coating Affects DBM Osteoinductivity In Vivo by Changing Inflammatory Responses.

PEGylation is a widely used modification in device coating or drug delivery by combing materials with poly(ethylene glycol) (PEG). In the present study, a well-established rat ectopic bone formation model was used to elucidate how PEGylated coating affects demineralized bone matrix (DBM) osteoinductivity in vivo by changing the inflammation events at the early phase of implantation. A range of cell-matrix interactions was characterized at the cellular and functional levels, including growth factor activity and kinetics, immune cell migration and activation, and bone formation in vivo. After 28 days, DBM's bone formation potential decreased in groups with increasing PEG concentration in the gelatin carrier. The increasing PEG concentration did not affect DBM's osteoinductive growth factor release or activity. However, increasing PEG cross-linking concentration resulted in decreased DBM-related early phase inflammatory reactions, reduced neutrophil infiltration, decreased coating material degradation, lowered the total number and active mast cells, and decreased CD80+ macrophage expression. Understanding and controlling cell-material responses may improve the design and development of functional medical devices.

From competency to dormancy: a 3D model to study cancer cells and drug responsiveness.

BACKGROUND: The heterogeneous and dynamic tumor microenvironment has significant impact on cancer cell proliferation, invasion, drug response, and is probably associated with entering dormancy and recurrence. However, these complex settings are hard to recapitulate in vitro. METHODS: In this study, we mimic different restriction forces that tumor cells are exposed to using a physiologically relevant 3D model with tunable mechanical stiffness. RESULTS: Breast cancer MDA-MB-231, colon cancer HCT-116 and pancreatic cancer CFPAC cells embedded in the stiffer gels exhibit a changed morphology and cluster formation, prolonged doubling time, and a slower metabolism rate, recapitulating the pathway from competency to dormancy. Altering environmental restriction allows them to re-enter and exit dormant conditions and change their sensitivities to drugs such as paclitaxol and gemcitabine. Cells surviving drug treatments can still regain competent growth and form tumors in vivo. CONCLUSION: We have successfully developed an in vitro 3D model to mimic the effects of matrix restriction on tumor cells and this high throughput model can be used to study tumor cellular functions and their drug responses in their different states. This all in one platform may aid effective drug development.

Acidosis and Formaldehyde Secretion as a Possible Pathway of Cancer Pain and Options for Improved Cancer Pain Control.

The prevalence of cancer pain in patients with cancer is high. The majority of efforts are spent on research in cancer treatment, but only a small fraction focuses on cancer pain. Pain in cancer patients, viewed predominantly as a secondary issue, is considered to be due to the destruction of tissues, compression of the nerves, inflammation, and secretion of biological mediators from the necrotic tumor mass. As a result, opioid drugs have remained as the primary pharmacological therapy for cancer pain for the past hundred years. This report reviews evidence that cancer pain may be produced by the metabolic effects of two byproducts of cancer-high acidity in the cancer microenvironment and the secretion of formaldehyde and its metabolites. We propose the research and development of therapeutic approaches for preemptive, short- and long-term management of cancer pain using available drugs or nutraceutical agents that can suppress or neutralize lactic acid production in combination with formaldehyde scavengers. We believe this approach may not only improve cancer pain control but may also enhance the quality of life for patients.

Tumor bioengineering using a transglutaminase crosslinked hydrogel.

Development of a physiologically relevant 3D model system for cancer research and drug development is a current challenge. We have adopted a 3D culture system based on a transglutaminase-crosslinked gelatin gel (Col-Tgel) to mimic the tumor 3D microenvironment. The system has several unique advantages over other alternatives including presenting cell-matrix interaction sites from collagen-derived peptides, geometry-initiated multicellular tumor spheroids, and metabolic gradients in the tumor microenvironment. Also it provides a controllable wide spectrum of gel stiffness for mechanical signals, and technical compatibility with imaging based screening due to its transparent properties. In addition, the Col-Tgel provides a cure-in-situ delivery vehicle for tumor xenograft formation in animals enhancing tumor cell uptake rate. Overall, this distinctive 3D system could offer a platform to more accurately mimic in vivo situations to study tumor formation and progression both in vitro and in vivo.

The synergetic effect of hydrogel stiffness and growth factor on osteogenic differentiation.

Cells respond to various chemical signals as well as environmental aspects of the extracellular matrix (ECM) that may alter cellular structures and functions. Hence, better understanding of the mechanical stimuli of the matrix is essential for creating an adjuvant material that mimics the physiological environment to support cell growth and differentiation, and control the release of the growth factor. In this study, we utilized the property of transglutaminase cross-linked gelatin (TG-Gel), where modification of the mechanical properties of TG-Gel can be easily achieved by tuning the concentration of gelatin. Modifying one or more of the material parameters will result in changes of the cellular responses, including different phenotype-specific gene expressions and functional differentiations. In this study, stiffer TG-Gels itself facilitated focal contact formation and osteogenic differentiation while soft TG-Gel promoted cell proliferation. We also evaluated the interactions between a stimulating factor (i.e. BMP-2) and matrix rigidity on osteogenesis both in vitro and in vivo. The results presented in this study suggest that the interactions of chemical and physical factors in ECM scaffolds may work synergistically to enhance bone regeneration.

Injectable gel graft for bone defect repair.

AIM: To examine the performance of an injectable gel graft made of transglutaminase (Tg)-crosslinked gelatin gel with BMP-2 (BMP-2-Tg-Gel) for bone defect repair in animal models. MATERIALS & METHODS: BMP-2 mixed with gelatin gel was crosslinked using Tg. The release of tethered BMP-2 through autocrine and paracrine pathways was demonstrated by using C2C12 and NIH 3T3 cells, respectively. BMP-2-Tg-Gel was injected into the induced cranial defect site. After 14 days, the sample was removed for x-ray imaging and histological evaluation. RESULTS: Our in vivo results demonstrated that the injectable Tg-Gel with its osteoconductivity and controllable BMP-2 activity induced bone formation in our rat models when tethered with BMP-2. CONCLUSION: Tg-Gel as an injectable functional bone graft may enable the use of minimally invasive surgical procedures to treat irregular-shaped bone defects. Furthermore, this novel approach is capable of incorporating and controlling the release of therapeutic agents that may advance the science of tissue regeneration.

The effects of heparin binding proteins in platelet releasate on bone formation.

Platelet releasates (PR) from platelet-rich plasma (PRP) preparations are often used as an adjuvant for tissue repair. However, many in vitro and in vivo published studies have shown inconsistent results since outcomes depended on the methods of PRP preparation, the dosage of PRP and wound repair models used. Among the 300 proteins released by platelets, a number of them have the ability to bind to heparin, a sulfated glycosaminoglycan well known for its anticoagulant activity. In this study, we focused on the heparin binding proteins in PR by testing PR with the heparin-heparin binding protein complexes, PR with slow releasing heparin binding proteins from agarose-bound heparin, and PR with the heparin binding proteins removed. The bone-forming effect of each of these PR as an adjuvant to demineralized bone matrix (DBM) was tested. When heparin was added to the PR preparation, DBM bone-forming potential was completely lost. However, when heparin was added as agarose-bound heparin and co-implanted with DBM, both alkaline phosphatase activity and areas of new bone formation increased. The study revealed that stripping off heparin binding proteins from PR did not affect ectopic DBM bone formation, whereas the controlled release of such proteins favors osteoinduction.

Enzymatic crosslinking and degradation of gelatin as a switch for bone morphogenetic protein-2 activity.

Current therapies for tissue regeneration rely on the presence or direct delivery of growth factors to sites of repair. Bone morphogenetic protein-2 (BMP-2), combined with a carrier (usually collagen), is clinically proven to induce new bone formation during spinal fusion and nonunion repair. However, due to BMP-2's short half-life and its diffusive properties, orders of magnitude above physiological levels are required to ensure effectiveness. In addition, a high dose of this multifunctional growth factor is known to induce adverse effects in patients. To circumvent these challenges, we proposed and tested a new approach for BMP-2 delivery, by controlling BMP activity via carrier binding and localized proteolysis. BMP-2 was covalently bound to gelatin through site-specific enzymatic crosslinking using a microbial transglutaminase. Binding of BMP-2 to gelatin can completely switch off BMP-2 activity, as evidenced by loss of its transdifferentiating ability toward C2C12 promyoblasts. When gelatin sequestered BMP-2 is incubated with either microbial collagenase or tissue-derived matrix metalloproteinases, BMP-2 activity is fully restored. The activity of released BMP-2 correlates with the protease activity in a dose- and time-dependent manner. This observation suggests a novel way of delivering BMP-2 and controlling its activity. This improved delivery method, which relies on a physiological feedback, should enhance the known potential of this and other growth factors for tissue repair and regeneration.