Xuezhong He

Migration of marrow stromal cells in response to sustained release of stromal-derived factor-1α from poly(lactide ethylene oxide fumarate) hydrogels

Stromal derived factor-1alpha (SDF-1alpha) is an important chemokine in stem cell trafficking and plays a critical role in the homing of bone marrow stromal (BMS) cells. However, its use in tissue regeneration is limited by its relatively short half-life and the time-dependent nature of cell homing to the site of injury. The objective of this work was to investigate the release characteristics of SDF-1alpha from degradable poly(lactide ethylene oxide fumarate) (PLEOF) hydrogels and to determine the effect of sustained release of SDF-1alpha on migration of BMS cells. Three PLEOF hydrogels with poly(l-lactide) (PLA) fractions of 6%, 9%, and 24% by weight were synthesized. After the addition of chemokine, the polymerizing mixture was crosslinked to produce SDF-1alpha loaded PLEOF hydrogels. The hydrogels were characterized with respect to sol fraction, water uptake, degradation, SDF-1alpha loading efficiency and release kinetics, and migration rate of bone marrow stromal (BMS) cells. The more hydrophilic hydrogels with 6% and 9% PLA fraction had a pronounced burst release followed by a period of sustained release by diffusion for 21 days. The more hydrophobic hydrogel with 24% PLA fraction had a less pronounced burst release and displayed a slow but constant release by diffusion between days 1 and 9 followed by a fast release by diffusion-degradation from days 9 to 18. The fraction of active SDF-1alpha released from 6%, 9%, and 24% hydrogels after 21 days was 34.3%, 32.3%, and 35.8%, respectively. The migration of BMS cells in response to time-released SDF-1alpha closely followed the protein release kinetics from the hydrogels. The biodegradable PLEOF hydrogel may potentially be useful as a delivery matrix for sustained release of SDF-1alpha in the proliferative phase of healing for recruitment of progenitor cells in tissue engineering applications.

Release characteristics and osteogenic activity of recombinant human bone morphogenetic protein-2 grafted to novel self-assembled poly(lactide-co-glycolide fumarate) nanoparticles

Functionalized biodegradable nanoparticles (NPs) provide reactive groups and large surface area for grafting recombinant human bone morphogenetic protein-2 (rhBMP-2) to reduce protein diffusion and maintain sufficient concentration for recruitment and differentiation of osteoprogenitor cells. The objective of this work was to investigate release characteristics and osteogenic activity of rhBMP-2, grafted to biodegradable NPs based on succinimide-terminated poly(lactide fumarate) (PLAF-NHS) and poly(lactide-co-glycolide fumarate) (PLGF-NHS) macromers. The release of rhBMP-2 from the NPs, measured by enzyme-linked immunosorbent assay, was linear with time in the first two weeks, and 24.70+/-1.30% and 48.7+/-0.7% of the protein grafted to PLGF-NHS and PLAF-NHS NPs, respectively, was released in the enzymatically active conformation after complete degradation/erosion of the NPs. After 14 days of incubation with bone marrow stromal (BMS) cells, rhBMP-2 grafted to PLAF-NHS and PLGF-NHS NPs was as effective in inducing mineralization as the native rhBMP-2 that was directly added to the cell culture media. At any incubation time, rhBMP-2 grafted to PLAF had the highest expression of osteopontin (OP) and osteocalcin (OC), followed by rhBMP-2 grafted to PLGF and rhBMP-2 directly added to media. Higher OP and OC expression for BMP-gPLAF and BMP-gPLGF groups may be related to other factors in the cascade of osteogenesis, such as differentiation of BMS cells to the vasculogenic lineage and formation of a vascularized/mineralized matrix.

Combined Effect of Osteopontin and BMP-2 Derived Peptides Grafted to an Adhesive Hydrogel on Osteogenic and Vasculogenic Differentiation of Marrow Stromal Cells

The objective of this work was to investigate the combined effect of grafting the peptide corresponding to amino acid residues 162-168 of osteopontin (OPD peptide) and the peptide corresponding to amino acid residues 73-92 of bone morphogenetic protein-2 (BMP peptide) to an RGD-conjugated inert hydrogel on osteogenic and vasculogenic differentiation of bone marrow stromal (BMS) cells. RGD-conjugated three-dimensional (3D) porous hydrogel scaffolds with well-defined cylindrical pore geometry were produced from sacrificial wax molds fabricated by fused deposition modeling rapid prototyping system. Propargyl acrylate and 4-pentenal were conjugated to the hydrogel for orthogonal grafting of BMP and OPD peptides by click reaction and oxime ligation, respectively. The OPD peptide was grafted by the reaction between aminooxy moiety of aminooxy-mPEG-OPD (mPEG = mini-poly(ethylene glycol)) and the aldehyde moiety in the hydrogel. The BMP peptide was grafted by the reaction between the azide moiety of Az-mPEG-BMP and the propargyl moiety in the hydrogel. The hydrogels seeded with BMS cells were characterized by biochemical, immunocytochemical, and mRNA analyses. Groups included RGD control hydrogel (RGD), RGD and BMP peptides without OPD (RGD+BMP), RGD and BMP peptides with mutant OPD (RGD+BMP+mOPD), and RGD and BMP peptides with OPD (RGD+BMP+OPD) grafted hydrogels. The extent of mineralization of RGD, RGD+BMP, RGD+BMP+mOPD, and RGD+BMP+OPD groups after 28 days was 650 ± 70, 990 ± 30, 850 ± 30, and 1150 ± 40 mg/(mg of DNA), respectively, indicating that the BMP and OPD peptides enhanced osteogenic differentiation of the BMS cells. The BMS cells seeded on RGD+BMP+OPD grafted hydrogels stained positive for vasculogenic markers α-SMA, PECAM-1, and VE-cadherin while the groups without OPD peptide (RGD+BMP and RGD+BMP+mOPD) stained only for α-SMA but not PECAM-1 or VE-cadherin. These results were consistent with the significantly higher PECAM-1 mRNA expression for RGD+BMP+OPD group after 21 and 28 days, compared to the groups without OPD. These findings suggest that the RGD+BMP+OPD peptides provide a favorable microenvironment for concurrent osteogenic and vasculogenic differentiation of progenitor marrow-derived cells.

Effect of CD44 Binding Peptide Conjugated to an Engineered Inert Matrix on Maintenance of Breast Cancer Stem Cells and Tumorsphere Formation

Introduction As cancer cells are affected by many factors in their microenvironment, a major challenge is to isolate the effect of a specific factor on cancer stem cells (CSCs) while keeping other factors unchanged. We have developed a synthetic inert 3D polyethylene glycol diacrylate (PEGDA) gel culture system as a unique tool to study the effect of microenvironmental factors on CSCs response. We have reported that CSCs formed in the inert PEGDA gel by encapsulation of breast cancer cells maintain their stemness within a certain range of gel stiffness. The objective was to investigate the effect of CD44 binding peptide (CD44BP) conjugated to the gel on the maintenance of breast CSCs. Methods 4T1 or MCF7 breast cancer cells were encapsulated in PEGDA gel with CD44BP conjugation. Control groups included dissolved CD44BP and the gel with mutant CD44BP conjugation. Tumorsphere size and density, and expression of CSC markers were determined after 9 days. For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined after 4 weeks. Effect of CD44BP conjugation on breast CSC maintenance was compared with integrin binding RGD peptide (IBP) and fibronectin-derived heparin binding peptide (FHBP). Results Conjugation of CD44BP to the gel inhibited breast tumorsphere formation in vitro and in vivo. The ability of the encapsulated cells to form tumorspheres in the peptide-conjugated gels correlated with the expression of CSC markers. Tumorsphere formation in vitro was enhanced by FHBP while it was abolished by IBP. Conclusion CD44BP and IBP conjugated to the gel abolished tumorsphere formation by encapsulated 4T1 cells while FHBP enhanced tumorsphere formation compared to cells in the gel without peptide. The PEGDA hydrogel culture system provides a novel tool to investigate the individual effect of factors in the microenvironment on CSC maintenance without interference of other factors.

Gelation Characteristics and Osteogenic Differentiation of Stromal Cells In Inert Hydrolytically Degradable Micellar Polyethylene Glycol Hydrogels

The use of poly(ethylene glycol) (PEG) hydrogels in tissue engineering is limited by their persistence in the site of regeneration. In an attempt to produce inert hydrolytically degradable PEG-based hydrogels, star (SPELA) poly(ethylene glycol-co-lactide) acrylate macromonomers with short lactide segments (<15 lactides per macromonomer) were synthesized. The SPELA hydrogel was characterized with respect to gelation time, modulus, water content, sol fraction, degradation, and osteogenic differentiation of encapsulated marrow stromal cells (MSCs). The properties of SPELA hydrogel were compared with those of the linear poly(ethylene glycol-co-lactide) acrylate (LPELA). The SPELA hydrogel had higher modulus, lower water content, and lower sol fraction than the LPELA. The shear modulus of SPELA hydrogel was 2.2 times higher than LPELA, whereas the sol fraction of SPELA hydrogel was 5 times lower than LPELA. The degradation of SPELA hydrogel depended strongly on the number of lactide monomers per macromonomer (nL) and showed a biphasic behavior. For example, as nL increased from 0 to 3.4, 6.4, 11.6, and 14.8, mass loss increased from 7 to 37, 80, 100% and then deceased to 87%, respectively, after 6 weeks of incubation. The addition of 3.4 lactides per macromonomer (<10 wt % dry macromonomer or <2 wt % swollen hydrogel) increased mass loss to 50% after 6 weeks. Molecular dynamic simulations demonstrated that the biphasic degradation behavior was related to aggregation and micelle formation of lactide monomers in the macromonomer in aqueous solution. MSCs encapsulated in SPELA hydrogel expressed osteogenic markers Dlx5, Runx2, osteopontin, and osteocalcin and formed a mineralized matrix. The expression of osteogenic markers and extent of mineralization was significantly higher when MSCs were encapsulated in SPELA hydrogel with the addition of bone morphogenetic protein-2 (BMP2). Results demonstrate that hydrolytically degradable PEG-based hydrogels are potentially useful as a delivery matrix for stem cells in regenerative medicine.

Drug Release Kinetics, Cell Uptake, and Tumor Toxicity of Hybrid VVVVVVKK Peptide-Assembled Polylactide Nanoparticles

An exciting approach to tumor delivery is encapsulation of the drug in self-assembled polymer-peptide nanoparticles. The objective of this work was to synthesize a conjugate of low molecular weight polylactide (LMW PLA) and V6K2 peptide and investigate self-assembly, drug release kinetics, cell uptake and toxicity, drug pharmacokinetics, and tumor cell invasion with Doxorubicin (DOX) or paclitaxel (PTX). The results for PLA-V6K2 self-assembled NPs were compared with those of polyethylene glycol stabilized PLA (PLA-EG) NPs. The size of PLA-V6K2 and PLA-EG NPs was 100 ± 20 and 130 ± 50 nm, respectively, with polydispersity index of 1.04 and 1.14. The encapsulation efficiency of DOX in PLA-V6K2 and PLA-EG NPs was 44 ± 9% and 55 ± 5%, respectively, and that of PTX was >90 for both NP types. The release of DOX and PTX from PLA-V6K2 was slower than that of PLA-EG, and the release rate was relatively constant with time. Based on molecular dynamic simulation, the less hydrophobic DOX was distributed in the lactide core as well as the peptide shell, while the hydrophobic PTX was localized mainly to the lactide core. PLA-V6K2 NPs had significantly higher cell uptake by 4T1 mouse breast carcinoma cells compared to PLA-EG NPs, which was attributed to the electrostatic interactions between the peptide and negatively charged moieties on the cell membrane. PLA-V6K2 NPs showed no toxicity to marrow stromal cells. DOX-loaded PLA-V6K2 NPs showed higher toxicity to 4T1 cells and the DNA damage response, and apoptosis was delayed compared to the free DOX. DOX or PTX encapsulated in PLA-V6K2 NPs significantly reduced invasion of 4T1 cells compared to those cells treated with the drug in PLA-EG NPs. Invasion of 4T1 cells treated with DOX in PLA-V6K2 and PLA-EG NPs was 5 ± 1% and 30 ± 5%, respectively, and that of PTX was 11 ± 2% and 40 ± 7%. The AUC of DOX in PLA-V6K2 NPs was 67% and 21% higher than those of free DOX and PLA-EG NPs, respectively. DOX-loaded PLA-V6K2 NPs injected in C3HeB/FeJ mice inoculated with MTCL syngeneic breast cancer cells displayed higher tumor toxicity than PLA-EG NPs and lower host toxicity than the free DOX. Cationic PLA-V6K2 NPs with higher tumor toxicity than the PLA-EG NPs are potentially useful in chemotherapy.

Solid-Phase Synthesis of Reactive Peptide Crosslinker by Selective Deprotection

An effective and simple strategy for preparing peptide crosslinkers is described. An MMP-13 degradable peptide QPQGLAK-NH(2) was prepared on the solid-phase using Fmoc chemistry. The peptide crosslinker was synthesized on-bead by the coupling reaction between acrylic acid and the amine groups of glutamine and lysine residues. The synthetic procedure employed the acid-labile Fmoc-Lys (Mtt)-OH and base-labile Fmoc-AA-OH derivatives. Selective deprotection, of -Mtt and -Fmoc groups on-bead, freed the amine end-groups on glutamine and lysine residues for coupling reaction with acrylic acid while maintaining the peptide attached to the resin. Subsequent cleavage from the resin yielded a peptide crosslinker with two unsaturated acrylate end-groups with high yield and purity. This method can be generally employed for the synthesis of a wide range of peptides with one or more reactive groups for grafting in the fabrication of biomimetic scaffolds in tissue engineering applications.

Effect of surface modification of nanofibres with glutamic acid peptide on calcium phosphate nucleation and osteogenic differentiation of marrow stromal cells

Biomineralization is mediated by extracellular matrix (ECM) proteins with amino acid sequences rich in glutamic acid. The objective of this study was to investigate the effect of calcium phosphate deposition on aligned nanofibres surface‐modified with a glutamic acid peptide on osteogenic differentiation of rat marrow stromal cells. Blend of EEGGC peptide (GLU) conjugated low molecular weight polylactide (PLA) and high molecular weight poly(lactide‐co‐glycolide) (PLGA) was electrospun to form aligned nanofibres (GLU‐NF). The GLU‐NF microsheets were incubated in a modified simulated body fluid for nucleation of calcium phosphate crystals on the fibre surface. To achieve a high calcium phosphate to fibre ratio, a layer‐by‐layer approach was used to improve diffusion of calcium and phosphate ions inside the microsheets. Based on dissipative particle dynamics simulation of PLGA/PLA–GLU fibres, > 80% of GLU peptide was localized to the fibre surface. Calcium phosphate to fibre ratios as high as 200%, between those of cancellous (160%) and cortical (310%) bone, was obtained with the layer‐by‐layer approach. The extent of osteogenic differentiation and mineralization of marrow stromal cells seeded on GLU‐NF microsheets was directly related to the amount of calcium phosphate deposition on the fibres prior to cell seeding. Expression of osteogenic markers osteopontin, alkaline phosphatase (ALP), osteocalcin and type 1 collagen increased gradually with calcium phosphate deposition on GLU‐NF microsheets. Results demonstrate that surface modification of aligned synthetic nanofibres with EEGGC peptide dramatically affects nucleation and growth of calcium phosphate crystals on the fibres leading to increased osteogenic differentiation of marrow stromal cells and mineralization. Copyright

Effect of Grafting BMP2 Derived Peptide to Nanoparticles on Osteogenic and Vasculogenic Expression of Stromal Cells

Bone morphogenetic protein‐2 (BMP2) plays a major role in initiating the cascade of osteogenesis. However, high doses of exogenous BMP2 coupled with diffusion away from the intended site cause adverse side‐effects. An alternative is to use biodegradable polymeric nanoparticles (NPs) grafted with peptides of the active domains of BMP2. NPs present a multivalent form of the peptide for stronger interaction with cell surface receptors, leading to a stronger activation of osteogenic signalling pathways. The objective of this work was to compare osteogenic activity of the BMP2 peptide (BMP2Pe), corresponding to residues 73–92 of BMP2 protein (BMP2Pr), grafted to biodegradable NPs with that of BMP2 protein (BMP2Pr). BMP2Pe was functionalized with a cysteine residue and grafted to poly(lactide fumarate) and poly(lactide‐co‐ethylene oxide fumarate) (PLAF/PLEOF) NPs via a thioether link. The calcium content of bone marrow stromal (BMS) cells cultured in osteogenic medium supplemented with BMP2 peptide/protein‐grafted NPs (BMP2Pe‐gNP and BMP2Pr‐gNP) was slightly higher than other BMP2‐treated groups, but all osteogenic groups showed similar levels of mineralization after 21 days. The expression pattern of master transcription factors Dlx5 and Runx2 indicated that BMP2 protein induced faster osteogenic signalling than the BMP peptide. The expression level of Osteopontin (OP), Osteocalcin (OC) and PECAM‐1 in the NP‐grafted BMP2 groups was significantly higher than those of ungrafted BMP2Pr and BMP2Pe groups, which may be due to a more effective presentation of the peptide/protein to cell surface receptors, thus leading to a stronger interaction of the peptide/protein with clustered cell surface receptors. Copyright

The role of filler-matrix interaction on viscoelastic response of biomimetic nanocomposite hydrogels

The effect of a glutamic acid (negatively charged) peptide (Glu6), which mimics the terminal region of the osteonectin glycoprotein of bone on the shear modulus of a synthetic hydorgel/apatite nanocomposite, was investigated. One end of the synthesized peptide was functionalized with an acrylate group (Ac-Glu6) to covalently attach the peptide to the hydrogel phase of the composite matrix. The addition of Ac-Glu6 to hydroxyapatite (HA) nanoparticles (50nm in size) resulted in significant reinforcement of the shear modulus of the nanocomposite (∼100% increase in elastic shear modulus). The reinforcement effect of the Glu6 peptide, a sequence in the terminal region of osteonectin, was modulated by the size of the apatite crystals. A molecular model is also proposed to demonstrate the role of polymer-apatite interaction in improving the viscoelastic behavior of the bone mimetic composite. The predictions of the model were compared with the measured dynamic shear modulus of the PLEOF hydrogel reinforced with HA nanoparticles. This predictive model provides a quantitative framework to optimize the properties of reinforced polymer nanocomposites as scaffolds for applications in tissue regeneration.