Citrate-based materials fuel human stem cells by metabonegenic regulation.

A comprehensive understanding of the key microenvironmental signals regulating bone regeneration is pivotal for the effective design of bioinspired orthopedic materials. Here, we identified citrate as an osteopromotive factor and revealed its metabonegenic role in mediating citrate metabolism and its downstream effects on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). Our studies show that extracellular citrate uptake through solute carrier family 13, member 5 (SLC13a5) supports osteogenic differentiation via regulation of energy-producing metabolic pathways, leading to elevated cell energy status that fuels the high metabolic demands of hMSC osteodifferentiation. We next identified citrate and phosphoserine (PSer) as a synergistic pair in polymeric design, exhibiting concerted action not only in metabonegenic potential for orthopedic regeneration but also in facile reactivity in a fluorescent system for materials tracking and imaging. We designed a citrate/phosphoserine-based photoluminescent biodegradable polymer (BPLP-PSer), which was fabricated into BPLP-PSer/hydroxyapatite composite microparticulate scaffolds that demonstrated significant improvements in bone regeneration and tissue response in rat femoral-condyle and cranial-defect models. We believe that the present study may inspire the development of new generations of biomimetic biomaterials that better recapitulate the metabolic microenvironments of stem cells to meet the dynamic needs of cellular growth, differentiation, and maturation for use in tissue engineering.

Development of Citrate-based Dual-Imaging Enabled Biodegradable Electroactive Polymers.

Increasing occurrences of degenerative diseases, defective tissues and severe cancers heighten the importance of advanced biomedical treatments, which in turn enhance the need for improved biomaterials with versatile theranostic functionalities yet using minimal design complexity. Leveraging the advantages of citrate chemistry, we developed a multifunctional citrate-based biomaterial platform with both imaging and therapeutic capabilities utilizing a facile and efficient one-pot synthesis. The resulting aniline tetramer doped biodegradable photoluminescent polymers (BPLPATs) not only possess programmable degradation profiles (<1 to >6 months) and mechanical strengths (~20 MPa to > 400 MPa), but also present a combination of intrinsic fluorescence, photoacoustic (PA) and electrical conductivity properties. BPLPAT nanoparticles are able to label cells for fluorescence imaging and perform deep tissue detection with PA imaging. Coupled with significant photothermal performance, BPLPAT nanoparticles demonstrate great potential for thermal treatment and in vivo real-time detection of cancers. Our results on BPLPAT scaffolds demonstrate three-dimensional (3D) high-spatial-resolution deep tissue PA imaging (23 mm), as well as promote growth and differentiation of PC-12 nerve cells. We envision that the biodegradable dual-imaging-enabled electroactive citrate-based biomaterial platform will expand the currently available theranostic material systems and open new avenues for diversified biomedical and biological applications via the demonstrated multi-functionality.

Immune Cell-Mediated Biodegradable Theranostic Nanoparticles for Melanoma Targeting and Drug Delivery.

Although tremendous efforts have been made on targeted drug delivery systems, current therapy outcomes still suffer from low circulating time and limited targeting efficiency. The integration of cell-mediated drug delivery and theranostic nanomedicine can potentially improve cancer management in both therapeutic and diagnostic applications. By taking advantage of innate immune cell's ability to target tumor cells, the authors develop a novel drug delivery system by using macrophages as both nanoparticle (NP) carriers and navigators to achieve cancer-specific drug delivery. Theranostic NPs are fabricated from a unique polymer, biodegradable photoluminescent poly (lactic acid) (BPLP-PLA), which possesses strong fluorescence, biodegradability, and cytocompatibility. In order to minimize the toxicity of cancer drugs to immune cells and other healthy cells, an anti-BRAF V600E mutant melanoma specific drug (PLX4032) is loaded into BPLP-PLA nanoparticles. Muramyl tripeptide is also conjugated onto the nanoparticles to improve the nanoparticle loading efficiency. The resulting nanoparticles are internalized within macrophages, which are tracked via the intrinsic fluorescence of BPLP-PLA. Macrophages carrying nanoparticles deliver drugs to melanoma cells via cell-cell binding. Pharmacological studies also indicate that the PLX4032 loaded nanoparticles effectively kill melanoma cells. The "self-powered" immune cell-mediated drug delivery system demonstrates a potentially significant advancement in targeted theranostic cancer nanotechnologies.

Cell adhesion and invasion inhibitory effect of an ovarian cancer targeting peptide selected via phage display in vivo.

Organ-specific metastasis is of great importance since most of the cancer deaths are caused by spread of the primary cancer to distant sites. Therefore, targeted anti-metastases therapies are needed to prevent cancer cells from metastasizing to different organs. The phage clone pc3-1 displaying peptide WSGPGVWGASVK selected by phage display had been identified which have high binding efficiency and remarkable cell specificity to SK-OV-3 cells. In the present work, the effects of selected cell-binding phage and cognate peptide on the cell adhesion and invasion of targeted cells were investigated. Results showed that the adhesive ability of SK-OV-3 to extracellular matrix was inhibited by pc3-1 and peptide WSGPGVWGASVK, and pc3-1 blocked SK-OV-3 cells attachment more effective than the cognate peptide. The peptide WSGPGVWGASVK suppressed the cell number of SK-OV-3 that attached to HUVECs monolayer up to 24% and could block the spreading of the attaching cells. Forthermore, the cognate peptide could inhibit the invasion of SK-OV-3 significantly. The number of invaded SK-OV-3 cells and invaded SK-OV-3-activated HUVECs pretreated with peptide WSGPGVWGASVK was decreased by 24.3% and 36.6%, respectively. All these results suggested that peptide WSGPGVWGASVK might possess anti-metastasis against SK-OV-3 cells.

A novel peptide specifically targeting ovarian cancer identified by in vivo phage display.

Discovery of peptide ligands that can target human ovarian cancer and deliver chemotherapeutics offers new opportunity for cancer therapy. The advent of phage-displayed peptide library facilitated the screening of such peptides. In vivo screening that set in a microanatomic and functional context was applied in our study, and a novel peptide WSGPGVWGASVK targeting ovarian cancer was isolated. The phage clone PC3-1 displaying peptide WSGPGVWGASVK can gain effective access to accumulate in the tumor sites after intravenous injection while reducing its accumulation in normal organs. Positive immunostaining of PC3-1 was located in both sites of tumor cells and tumor blood vessels, which resulted in a diffuse binding pattern through the tumor. In vitro study results confirmed the capability of peptide WSGPGVWGASVK binding to and being internalized by both tumor cells and angiogenic endothelial cells. Flow cytometry analysis revealed that the peptide bound to SKOV3 cells with Kd value of 5.43 ± 0.4 µM. Taken together, it suggested that peptide WSGPGVWGASVK is a lead candidate for delivering therapeutics to penetrate into tumors.

A specific cell-penetrating peptide induces apoptosis in SKOV3 cells by down-regulation of Bcl-2.

Peptides are emerging as pharmaceutical agents in cancer therapy. The peptide, TLSGAFELSRDK (TLS) is a targeting ligand that can specifically triggers cellular uptake by binding to SKOV3 cells. Cell surface proteins and the C-terminal basic residues of the TLS are required for effective cell penetration, and the uptake process is energy-dependent. It inhibited the proliferation of SKOV3 cells and induced early-stage apoptosis by down-regulation of Bcl-2 expression mediated through a caspase-dependent pathway. The synergistic anti-proliferative effects of the peptide TLS and doxorubicin on SKOV3 cells were further investigated. Taken together, TLS, acting as a combination of a targeted ligand and a therapeutic agent, was a promising candidate for the development of peptide-based therapies in ovarian cancer.

Bioinspired Super-Strong Aqueous Synthetic Tissue Adhesives.

Existing tissue adhesives and sealants are far from satisfactory when applied on wet and dynamic tissues. Herein, we report a strategy for designing biodegradable super-strong aqueous glue (B-Seal) for surgical uses inspired by an English ivy adhesion strategy and a cement particle packing theory. B-Seal is a fast-gelling, super-strong, and elastic adhesive sealant composed of injectable water-borne biodegradable polyurethane (WPU) nanodispersions with mismatched particle sizes and counterions in its A-B formulation. B-Seal showed 24-fold greater burst pressure than DuraSeal®, 138-fold greater T-pull adhesive strength than fibrin glue, and 16-fold greater lap shear strength than fibrin glue. In vivo evaluation on a rat cerebrospinal fluid (CSF) rhinorrhea model and a porcine craniotomy model validated the safety and efficacy of B-Seal for effective CSF leak prevention and dura repair. The plant-inspired adhesion strategy combined with particle packing theory represents a new direction of designing the next-generation wet tissue adhesives for surgeries.

In vitro screening of ovarian tumor specific peptides from a phage display peptide library.

To develop more biomarkers for diagnosis and therapy of ovarian cancer, a 12-mer phage display library was used to isolate peptides that bound specifically to the human ovarian tumor cell line SK-OV-3. After five rounds of in vitro screening, the recovery rate of phages showed a 69-fold increase over the first round of washings and a group of phage clones capable of binding to SK-OV-3 cells were obtained. A phage clone named Z1 with high affinity and specificity to SK-OV-3 cells was identified in vitro. More importantly, the synthetic biotin-labeled peptide, ZP1 (=SVSVGMKPSPRP), which corresponded to the sequence of the inserted fragment of Z1, demonstrated a high specificity to SK-OV-3 cells especially when compared to other cell lines (A2780 and 3T3). ZP1 might therefore be a biomarker for targeting drug delivery in ovarian cancer therapy.

Antimicrobial hydroxyapatite and its composites for the repair of infected femoral condyle.

Orthopedic implant-associated infection constitutes one of the most devastating and challenging symptoms in the clinic. Implants without antimicrobial properties may become the harbourage for microbial colonization and biofilm formation, thus hindering normal bone regeneration processes. We had previously developed tannin modified HA (THA) as well as silver and tannin modified hydroxyapatite (HA) (Ag-THA) via a facile one-step and scalable process, and proven their antimicrobial performance in vitro. Herein, by compositing with non-antimicrobial polyurethane (PU), the in vivo anti-bacterial activity, osteoconductivity and osteoinductivity of PU/Ag-THA composite were investigated using an infected femoral condyle defect model on rat. PU/Ag-THA exhibited excellent in vivo antimicrobial activity, with the calculated bacteria fraction being reduced to lower than 3% at week 12 post operation. Meanwhile, PU/Ag-THA is also promising for bone regeneration under the bacteria challenge, evidenced by a final bone mineral density (BMD) ~0.6 times higher than that of the blank control at week 12. A continuous increase in BMD over time was observed in the PU/Ag-THA group, but not in the blank control and its non- or weak-antimicrobial counterparts (PU/HA and PU/THA), in which the growth rate of BMD declined after 8 weeks of operation. The enhanced osteoinductivity of PU/Ag-THA relative to blank control, PU/HA and PU/THA was also confirmed by the Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) immunohistochemical staining. The above findings suggest that antimicrobial Ag-THA may serve as a promising and easy-to-produce antimicrobial mineral for the development of antimicrobial orthopedic composite implants to address the challenges in orthopedic surgeries, especially where infection may become a challenging condition to treat.

In vivo study of polyurethane and tannin-modified hydroxyapatite composites for calvarial regeneration.

Biomaterial mediated bone regeneration is an attractive strategy for bone defect treatment. Organic/inorganic composites have been well established as effective bone graft. Here, the bone regenerative effect of the composites made from tannic acid (TA) modified hydroxyapatite (HA) (THA) or TA & silver nanoparticles (Ag NPs) modified HA (Ag-THA) and polyurethane (PU) was evaluated on critical-sized calvarial defects in rats. The in vivo study indicates that PU/THA and PU/Ag-THA scaffolds exhibited acceptable biocompatibility and induced significantly enhanced bone mineral densities comparing with the blank control (CON) group as well as PU/HA group. The inclusion of TA on HA brought the composites with enhanced osteogenesis and angiogenesis, evidenced by osteocalcin (OCN) and vascular endothelial growth factor (VEGF) immunohistochemical staining. Tartrate resistant acid phosphatase (TRAP) staining showed high osteoclast activity along with osteogenesis, especially in PU/THA and PU/Ag-THA groups. However, further introduction of Ag NPs on HA depressed the angiogenesis of the composites, leading to even lower VEGF expression than that of CON group. This study once more proved that THA can serve as a better bone composite component that pure HA and can promote osteogenesis and angiogenesis. While, the introduction of antimicrobial Ag NPs on HA need to be controlled in some extent not to affect the angiogenesis of the composites.

Enabling biodegradable functional biomaterials for the management of neurological disorders.

An increasing number of patients are being diagnosed with neurological diseases, but are rarely cured because of the lack of curative therapeutic approaches. This situation creates an urgent clinical need to develop effective diagnosis and treatment strategies for repair and regeneration of injured or diseased neural tissues. In this regard, biodegradable functional biomaterials provide promising solutions to meet this demand owing to their unique responsiveness to external stimulation fields, which enable neuro-imaging, neuro-sensing, specific targeting, hyperthermia treatment, controlled drug delivery, and nerve regeneration. This review discusses recent progress in the research and development of biodegradable functional biomaterials including electroactive biomaterials, magnetic materials and photoactive biomaterials for the management of neurological disorders with emphasis on their applications in bioimaging (photoacoustic imaging, MRI and fluorescence imaging), biosensing (electrochemical sensing, magnetic sensing and opical sensing), and therapy strategies (drug delivery, hyperthermia treatment, and tissue engineering). It is expected that this review will provide an insightful discussion on the roles of biodegradable functional biomaterials in the diagnosis and treatment of neurological diseases, and lead to innovations for the design and development of the next generation biodegradable functional biomaterials.

Biomaterial-Based Metabolic Regulation in Regenerative Engineering.

Recent advances in cell metabolism studies have deepened the appreciation of the role of metabolic regulation in influencing cell behavior during differentiation, angiogenesis, and immune response in the regenerative engineering scenarios. However, the understanding of whether the intracellular metabolic pathways could be influenced by material-derived cues remains limited, although it is now well appreciated that material cues modulate cell functions. Here, an overview of how the regulation of different aspect of cell metabolism, including energy homeostasis, oxygen homeostasis, and redox homeostasis could contribute to modulation of cell function is provided. Furthermore, recent evidence demonstrating how material cues, including the release of inherent metabolic factors (e.g., ions, regulatory metabolites, and oxygen), tuning of the biochemical cues (e.g., inherent antioxidant properties, cell adhesivity, and chemical composition of nanomaterials), and changing in biophysical cues (topography and surface stiffness), may impact cell metabolism toward modulated cell behavior are discussed. Based on the resurgence of interest in cell metabolism and metabolic regulation, further development of biomaterials enabling metabolic regulation toward dictating cell function is poised to have substantial implications for regenerative engineering.

Development of osteopromotive poly (octamethylene citrate glycerophosphate) for enhanced bone regeneration.

The design and development of bioactive materials that are inherently conducive for osteointegration and bone regeneration with tunable mechanical properties and degradation remains a challenge. Herein, we report the development of a new class of citrate-based materials with glycerophosphate salts, ß-glycerophosphate disodium (ß-GP-Na) and glycerophosphate calcium (GP-Ca), incorporated through a simple and convenient one-pot condensation reaction, which might address the above challenge in the search of suitable orthopedic biomaterials. Tensile strength of the resultant poly (octamethylene citrate glycerophosphate), POC-ßGP-Na and POC-GP-Ca, was as high as 28.2 ±â€¯2.44  MPa and 22.76 ±â€¯1.06  MPa, respectively. The initial modulus ranged from 5.28 ±â€¯0.56  MPa to 256.44 ±â€¯22.88  MPa. The mechanical properties and degradation rate of POC-GP could be controlled by varying the type of salts, and the feeding ratio of salts introduced. Particularly, POC-GP-Ca demonstrated better cytocompatibility and the corresponding composite POC-GP-Ca/hydroxyapatite (HA) also elicited improved osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro, as compared to POC-ßGP-Na/HA and POC/HA. The superior in-vivo performance of POC-GP-Ca/HA microparticle scaffolds in promoting bone regeneration over POC-ßGP-Na/HA and POC/HA was further confirmed in a rabbit femoral condyle defect model. Taken together, the tunability of mechanical properties and degradation rates, together with the osteopromotive nature of POC-GP polymers make these materials, especially POC-GP-Ca well suited for bone tissue engineering applications. STATEMENT OF SIGNIFICANCE: The design and development of bioactive materials that are inherently conducive for osteointegration and bone regeneration with tunable mechanical properties and degradation remains a challenge. Herein, we report the development of a new class of citrate-based materials with glycerophosphate salts, ß-glycerophosphate disodium (ß-GPNa) and glycerophosphate calcium (GPCa), incorporated through a simple and convenient one-pot condensation reaction. The resultant POC-GP polymers showed significantly improved mechanical property and tunable degradation rate. Within the formulation investigated, POC-GPCa/HA composite further demonstrated better bioactivity in favoring osteogenic differentiation of hMSCs in vitro and promoted bone regeneration in rabbit femoral condyle defects. The development of POC-GP expands the repertoire of the well-recognized citrate-based biomaterials to meet the ever-increasing needs for functional biomaterials in tissue engineering and other biomedical applications.

Citrate chemistry and biology for biomaterials design.

Leveraging the multifunctional nature of citrate in chemistry and inspired by its important role in biological tissues, a class of highly versatile and functional citrate-based materials (CBBs) has been developed via facile and cost-effective polycondensation. CBBs exhibiting tunable mechanical properties and degradation rates, together with excellent biocompatibility and processability, have been successfully applied in vitro and in vivo for applications ranging from soft to hard tissue regeneration, as well as for nanomedicine designs. We summarize in the review, chemistry considerations for CBBs design to tune polymer properties and to introduce functionality with a focus on the most recent advances, biological functions of citrate in native tissues with the new notion of degradation products as cell modulator highlighted, and the applications of CBBs in wound healing, nanomedicine, orthopedic, cardiovascular, nerve and bladder tissue engineering. Given the expansive evidence for citrate's potential in biology and biomaterial science outlined in this review, it is expected that citrate based materials will continue to play an important role in regenerative engineering.

In vitro cytocompatibility evaluation of poly(octamethylene citrate) monomers toward their use in orthopedic regenerative engineering.

Citrate based polymer poly(octamethylene citrate) (POC) has shown promise when formulated into composite material containing up to 65 wt% hydroxylapatite (HA) for orthopedic applications. Despite significant research into POC, insufficient information about the biocompatibility of the monomers 1,8-Octanediol and Citrate used in its synthesis is available. Herein, we investigated the acute cytotoxicity, immune response, and long-term functionality of both monomers. Our results showed a cell-type dependent cytotoxicity of the two monomers: 1,8-Octanediol induced less acute toxicity to 3T3 fibroblasts than Citrate while presenting comparable cytotoxicity to MG63 osteoblast-like cells; however, Citrate demonstrated enhanced compatibility with hMSCs compared to 1,8-Octanediol. The critical cytotoxic concentration values EC30 and EC50, standard for comparing cytotoxicity of chemicals, were also provided. Additionally, Citrate showed slower and less inhibitory effects on long-term hMSC cell proliferation compared with 1,8-Octanediol. Furthermore, osteogenic differentiation of hMSCs exposure to Citrate resulted in less inhibitory effect on alkaline phosphatase (ALP) production. Neither monomer triggered undesired pro-inflammatory responses. In combination with diffusion model analysis of monomer release from cylindrical implants, based on which the maximum concentration of monomers in contact with bone tissue was estimated to be 2.2 × 10-4 mmol/L, far lower than the critical cytotoxic concentrations as well as the 1,8-Octanediol concentration (0.4 mg/mL or 2.7 mmol/L) affecting hMSCs differentiation, we provide strong evidence for the cytocompatibility of the two monomers degraded from citrate-based composites in the orthopedic setting.

Inhibiting the motility and invasion of cancer cells by biomineralization.

Many kinds of cancer are difficult to treat because of their highly metastatic abilities. Thus, seeking new anticancer drugs or therapy strategies, which could reduce the motility of cancer cells or inhibit the migration and invasion of the cells, is an urgent affair. Several recent reports suggest various techniques (such as layer-by-layer assembly and biomimetic mineralization) aimed to functionalize human cells and microbial with polyelectrolytes, nanoparticles, or mineral coatings. Inspired by these studies, an artificial mineral shell could be formed to enclose cancer cells under the regulation of SIBLINGs-like proteins. Consequently, the connection between the cancer cell and substrate would be interfered or inhibited. Therefore, the motility of cancer cells would be weakened or inhibited due to the restriction of the artificial mineral shell. This hypothetical strategy might be as a new concept for cancer therapy.

Synthesis and cellular compatibility of biomineralized Fe3O4 nanoparticles in tumor cells targeting peptides.

Fe3O4 nanoparticles (NPs) coated with WSG-peptide were prepared via a facile biomineralization technique at room temperature. The concentration of the peptides and the mixing time could substantially influence the morphology of as-prepared particles. The saturation magnetization of WSG-coated Fe3O4 particles were 35.92 emu/g, slightly higher than that of Fe3O4 without WSG peptides. Cell viability assay revealed that WSG-coated Fe3O4 particles had a good cellular compatibility. In addition, compared with Fe3O4 NPs, the mineralized Fe3O4 NPs coated with WSG peptides could more easily assemble into the cancer cell, indicating that the WSG-Fe3O4 NPs possess cancer targeting property. Thus, the WSG-coated Fe3O4 NPs could be used in cancer diagnosis and treatment fields.