Intervertebral Disc Regeneration Using Stem Cell/Growth Factor-Loaded Porous Particles with a Leaf-Stacked Structure.

Although biological therapies based on growth factors and transplanted cells have demonstrated some positive outcomes for intervertebral disc (IVD) regeneration, repeated injection of growth factors and cell leakage from the injection site remain considerable challenges for human therapeutic use. Herein, we prepare human bone marrow-derived mesenchymal stem cells (hBMSCs) and transforming growth factor-ß3 (TGF-ß3)-loaded porous particles with a unique leaf-stack structural morphology (LSS particles) as a combination bioactive delivery matrix for degenerated IVD. The LSS particles are fabricated with clinically acceptable biomaterials (polycaprolactone and tetraglycol) and procedures (simple heating and cooling). The LSS particles allow sustained release of TGF-ß3 for 18 days and stable cell adhesiveness without additional modifications of the particles. On the basis of in vitro and in vivo studies, it was observed that the hBMSCs/TGF-ß3-loaded LSS particles can provide a suitable milieu for chondrogenic differentiation of hBMSCs and effectively induce IVD regeneration in a beagle dog model. Thus, therapeutically loaded LSS particles offer the promise of an effective bioactive delivery system for regeneration of various tissues including IVD.

Bladder Regeneration Using a Polycaprolactone Scaffold with a Gradient Structure and Growth Factors in a Partially Cystectomized Rat Model.

BACKGROUND: Tissue engineering can be used for bladder augmentation. However, conventional scaffolds result in fibrosis and graft shrinkage. This study applied an alternative polycaprolactone (PCL)-based scaffold (diameter = 5 mm) with a noble gradient structure and growth factors (GFs) (epidermal growth factor, vascular endothelial growth factor, and basic fibroblast growth factor) to enhance bladder tissue regeneration in a rat model. METHODS: Partially excised urinary bladders of 5-week-old male Slc:SD rats were reconstructed with the scaffold (scaffold group) or the scaffold combined with GFs (GF group) and compared with sham-operated (control group) and untreated rats (partial cystectomy group). Evaluations of bladder volume, histology, immunohistochemistry (IHC), and molecular markers were performed at 4, 8, and 12 weeks after operation. RESULTS: The bladder volumes of the scaffold and GF group recovered to the normal range, and those of the GF group showed more enhanced augmentation. Histological evaluations revealed that the GF group showed more organized urothelial lining, dense extracellular matrix, frequent angiogenesis, and enhanced smooth muscle bundle regeneration than the scaffold group. IHC for α-smooth muscle actin, pan-cytokeratin, α-bungarotoxin, and CD8 revealed that the GF group showed high formation of smooth muscle, blood vessel, urothelium, neuromuscular junction and low immunogenicity. Concordantly, real-time polymerase chain reaction experiments revealed that the GF group showed a higher expression of transcripts associated with smooth muscle and urothelial differentiation. In a 6-month in vivo safety analysis, the GF group showed normal histology. CONCLUSION: This study showed that a PCL scaffold with a gradient structure incorporating GFs improved bladder regeneration functionally and histologically.

Parthenolide Has Negative Effects on In Vitro Enhanced Osteogenic Phenotypes by Inflammatory Cytokine TNF-α via Inhibiting JNK Signaling.

Nuclear factor kappa B (NF-κB) regulates inflammatory gene expression and represents a likely target for novel disease treatment approaches, including skeletal disorders. Several plant-derived sesquiterpene lactones can inhibit the activation of NF-κB. Parthenolide (PTL) is an abundant sesquiterpene lactone, found in Mexican Indian Asteraceae family plants, with reported anti-inflammatory activity, through the inhibition of a common step in the NF-κB activation pathway. This study examined the effects of PTL on the enhanced, in vitro, osteogenic phenotypes of human periosteum-derived cells (hPDCs), mediated by the inflammatory cytokine tumor necrosis factor (TNF)-α. PTL had no significant effects on hPDC viability or osteoblastic activities, whereas TNF-α had positive effects on the in vitro osteoblastic differentiation of hPDCs. c-Jun N-terminal kinase (JNK) signaling played an important role in the enhanced osteoblastic differentiation of TNF-α-treated hPDCs. Treatment with 1 µM PTL did not affect TNF-α-treated hPDCs; however, 5 and 10 µM PTL treatment decreased the histochemical detection and activity of alkaline phosphatase (ALP), alizarin red-positive mineralization, and the expression of ALP and osteocalcin mRNA. JNK phosphorylation decreased significantly in TNF-α-treated hPDCs pretreated with PTL. These results suggested that PTL exerts negative effects on the increased osteoblastic differentiation of TNF-α-treated hPDCs by inhibiting JNK signaling.

Oxygen-Releasing Microparticles for Cell Survival and Differentiation Ability under Hypoxia for Effective Bone Regeneration.

Sufficient oxygen delivery into tissue-engineered three-dimensional (3D) scaffolds to produce clinically applicable tissues/organs remains a challenge for researchers and clinicians. One potential strategy to overcome this limitation is the use of an oxygen releasing scaffold. In the present study, we prepared hollow microparticles (HPs) loaded with an emulsion of the oxygen carrier perfluorooctane (PFO; PFO-HPs) for the timely supply of oxygen to surrounding cells. These PFO-HPs prolonged the survival and preserved the osteogenic differentiation potency of human periosteal-derived cells ( hPDCs) under hypoxia. hPDCs seeded onto PFO-HPs formed new bone at a faster rate and with a higher bone density than hPDCs seeded onto phosphate buffered saline-loaded control HPs. These findings suggest that PFO-HPs provide a suitable environment for the survival and maintenance of differentiation ability of hPDCs at bony defects without vascular networks until new blood vessel ingrowth occurs, thus enhancing bone regeneration. PFO-HPs are a promising system for effective delivery of various functional cells, including stem cells and progenitor cells, to regenerate damaged tissues/organs.

Development of Porous Beads to Provide Regulated BMP-2 Stimulation for Varying Durations: In Vitro and In Vivo Studies for Bone Regeneration.

It is commonly accepted that the sustained release of bone morphogenetic protein-2 (BMP-2) can enhance bone regeneration and minimize its safety issues. However, little is known regarding the appropriate duration of BMP-2 stimulation for sufficient osteogenic differentiation and new bone formation because of the short half-life of BMP-2 in the physiological environment and the lack of a well-defined delivery matrix that can regulate the release period of BMP-2. In this study, we prepared porous poly(lactic-co-glycolic acid) (PLGA) beads with different surface pore sizes that can regulate the release period of BMP-2 (i.e., 7, 17, and 30 days) while providing the BMP-2 concentration required for bone regeneration. Our findings in both in vitro cell culture and in vivo animal studies using these BMP-2-loaded beads demonstrate that release of BMP-2 within 7 days affects only the initial differentiation of human periosteum-derived cells (hPDCs) and does not significantly enhance their subsequent differentiation into mature functional cells. However, extending the duration of BMP-2 stimulation over 17 days can provide a suitable environment for osteogenic differentiation of hPDCs and new bone formation.

Functional and histological evidence for the targeted therapy using biocompatible polycaprolactone beads and autologous myoblasts in a dog model of fecal incontinence.

BACKGROUND: Injection of bulking agents into the anal canal is limited by several factors, including biological resorption, particle migration, and ongoing degradation of the injected bulking agent. OBJECTIVE: We investigated whether an injection of polycaprolactone beads containing autologous myoblasts could improve sphincter function in a dog model of fecal incontinence. DESIGN: The control sham surgery group underwent skin incision around the anal sphincter (n = 5). Fecal incontinence was induced by resecting 25% of the posterior internal/external anal sphincter in another 10 dogs. After 1 month of sphincter injury, dogs were then treated with (n = 5) or without (n = 5) polycaprolactone beads containing PKH-26-labeled autologous myoblasts. SETTING: This study was conducted at the department of surgery in collaboration with the department of advanced materials. OUTCOME MEASURES: Three months after injection treatment, the resting and contractile pressure differences of the anal sphincter were compared, and histopathological studies were performed. RESULTS: The anal pressures in untreated dogs were significantly lower than those in the sham surgery group (p < 0.05). The resting and contractile pressure differences were higher in treated dogs than in untreated dogs (resting pressure difference: 0.7 ± 0.5 vs -0.6 ± 0.8 mmHg; coefficient of the difference in recovery rate, 0.38; 95% CI, 0.15-0.61, p = 0.001; contractile pressure difference: 1.1 ± 4.2 vs -3.9 ± 2.6 mmHg; coefficient, 1.63; 95% CI, 0.55-2.71, p = 0.003). Immunofluorescent staining confirmed that the myoblasts had differentiated and synthesized myosin heavy chain, as observed in vitro. LIMITATIONS: This study was limited by the lack of comparison of injecting beads containing autologous myoblasts with injecting myoblasts alone. CONCLUSION: This study shows that an injection of polycaprolactone beads containing autologous myoblasts may improve anal sphincter function in an animal model of fecal incontinence.

Coadministration of basic fibroblast growth factor-loaded polycaprolactone beads and autologous myoblasts in a dog model of fecal incontinence.

PURPOSE: Basic fibroblastic growth factor (bFGF), a member of the heparin-binding growth factor family, regulates muscle differentiation. We investigated whether coadministration of autologous myoblasts and bFGF-loaded polycaprolactone beads could improve sphincter recovery in a dog model of fecal incontinence (FI). METHODS: FI was induced by resecting 25% of the posterior anal sphincter in ten mongrel dogs. One month later, the dogs were randomized to receive either PKH-26-labeled autologous myoblasts alone (M group, five dogs) or autologous myoblasts and bFGF-loaded polycaprolactone beads (MBG group, five dogs). The outcomes included anal manometry, compound muscle action potentials (CMAPs) of the pudendal nerve, and histology. RESULTS: The increase in anal contractile pressure over 3 months was significantly greater in the MBG group (from 4.85 to 6.83 mmHg) than that in the M group (from 4.94 to 4.25 mmHg), with a coefficient for the difference in recovery rate of 2.672 (95% confidence interval [CI] 0.962 to 4.373, p = 0.002). The change in the CMAP amplitude was also significantly greater in the MBG group (from 0.59 to 1.56 mV) than that in the M group (from 0.81 to 0.67 mV) (coefficient 1.114, 95% CI 0.43 to 1.80, p = 0.001). Labeled cells were detected in 2/5 (40%) and 5/5 (100%) dogs in the M and MBG groups, respectively. CONCLUSION: Coadministration of bFGF-loaded PCL beads and autologous myoblasts improved the recovery of sphincter function in a dog model of FI and had better outcomes than cell-based therapy alone.

FOXO1 Is Involved in the Effects of Cigarette Smoke Extract on Osteoblastic Differentiation of Cultured Human Periosteum-derived Cells.

Cigarette smoke is associated with delayed fracture healing, alterations in mineral content, and osteoporosis, however, its effects on osteoblastic differentiation of osteoprogenitor cells are not fully understood. In the present study, we examined the effects of cigarette smoke extract (CSE) on osteoblastic differentiation of cultured human periosteum-derived cells. We found that CSE inhibited alkaline phosphatase (ALP) activity, mineralization and Runx2 transactivation of the periosteum-derived cells. Nucleofection of RUNX2 into the periosteum-derived cells increased expression of endogenous osteocalcin (OC) and ALP genes in osteogenic induction medium and increased OC expression in non-osteogenic medium. Treatment of the periosteum-derived cells with CSE resulted in decreased phosphorylation of AKT and forkhead box protein O1 (FOXO1). The AKT phosphorylation-resistant mutant, FOXO1-A3, inhibited transcriptional activity of RUNX2 in the periosteum-derived cells. The current study suggests one mechanism by which CSE exposure leads to inhibition of osteoblastic differentiation of cultured human periosteum-derived cells.

Dual growth factor-loaded in situ gel-forming bulking agent: passive and bioactive effects for the treatment of urinary incontinence.

Stress urinary incontinence (SUI) is one of the major medical problems for adult females and has a devastating effect on their quality of life. The major cause of the development of the SUI is dysfunction of the urethral supporting tissues as a result of aging and childbirth. In this study, in situ gel-forming bulking agent loaded with dual growth factors, nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), was fabricated. The bulking agent consisted of three components; (i) polycaprolactone (PCL) beads, (ii) bFGF-loaded nanogels, and (iii) NGF-loaded in situ gel forming solution. The bulking agent can provide an initial passive bulking effect (from the PCL beads) and regenerate malfunctioning tissues around the urethra (from the sequential and continuous release of growth factors from the hydrogel) for the effective treatment of SUI. The PCL beads were located stably at the applied urethra site (urinary incontinent SD rat) without migration to provide a passive bulking effect. The sequential release of the growth factors (NGF within a week and bFGF for more than 4 weeks) from the bulking agent provided regeneration of damaged nerve and smooth muscle, and thus enhanced biological function around the urethra. From the findings, we suggest that dual growth factor (NGF and bFGF)-loaded in situ gel-forming bulking agent may be a promising injectable bioactive system for the treatment for SUI.

Human Urine-derived Stem Cells Seeded Surface Modified Composite Scaffold Grafts for Bladder Reconstruction in a Rat Model.

We conducted this study to investigate the synergistic effect of human urine-derived stem cells (USCs) and surface modified composite scaffold for bladder reconstruction in a rat model. The composite scaffold (Polycaprolactone/Pluronic F127/3 wt% bladder submucosa matrix) was fabricated using an immersion precipitation method, and heparin was immobilized on the surface via covalent conjugation. Basic fibroblast growth factor (bFGF) was loaded onto the heparin-immobilized scaffold by a simple dipping method. In maximal bladder capacity and compliance analysis at 8 weeks post operation, the USCs-scaffold(heparin-bFGF) group showed significant functional improvement (2.34 ± 0.25 mL and 55.09 ± 11.81 µL/cm H2O) compared to the other groups (2.60 ± 0.23 mL and 56.14 ± 9.00 µL/cm H2O for the control group, 1.46 ± 0.18 mL and 34.27 ± 4.42 µL/cm H2O for the partial cystectomy group, 1.76 ± 0.22 mL and 35.62 ± 6.69 µL/cm H2O for the scaffold group, and 1.92 ± 0.29 mL and 40.74 ± 7.88 µL/cm H2O for the scaffold(heparin-bFGF) group, respectively). In histological and immunohistochemical analysis, the USC-scaffold(heparin-bFGF) group showed pronounced, well-differentiated, and organized smooth muscle bundle formation, a multi-layered and pan-cytokeratin-positive urothelium, and high condensation of submucosal area. The USCs seeded scaffold(heparin-bFGF) exhibits significantly increased bladder capacity, compliance, regeneration of smooth muscle tissue, multi-layered urothelium, and condensed submucosa layers at the in vivo study.

Platelet-Rich Plasma-Embedded Porous Polycaprolactone Film with a Large Surface Area for Effective Hemostasis.

BACKGROUND: Uncontrollable and widespread bleeding caused by surgery or sudden accidents can lead to death if not treated with appropriate hemostasis. To prevent excessive life-threatening bleeding, various hemostatic agents based on polymeric biomaterials with various additives for accelerated blood coagulation have been adopted in clinical fields. In particular, platelet-rich plasma (PRP), which contains many blood coagulation factors that can accelerate blood clot formation, is considered as one of the most effective hemostatic additives. METHODS: We investigated a PRP-embedded porous film using discarded (expired) PRP and a film with a leaf-stacked structure (FLSS), as a hemostatic agent to induce rapid hemostasis. The film, which contained an LSS on one side (PCL-FLSS), was fabricated by a simple heating-cooling technique using tetraglycol and polycaprolactone (PCL) film. Activated PRP was obtained by the thawing of frozen PRP at the end of its expiration date (the platelet cell membrane is disrupted during the freezing and thawing of PRP, thus releasing various coagulation factors) and embedded in the PCL-FLSS (PRP-FLSS). RESULTS: From in vitro and in vivo experiments using a rat hepatic bleeding model, it was recognized that PRP-FLSS is not only biocompatible but also significantly accelerates blood clotting and thus prevents rapid bleeding, probably due to a synergistic effect of the sufficient supply of various blood coagulants from activated PRP embedded in the LSS layer and the large surface area of the LSS itself. CONCLUSION: The study suggests that PRP-FLSS, a combination of a porous polymer matrix with a unique morphology and discarded biofunctional resources, can be an advanced hemostatic agent as well as an upcycling platform to avoid the waste of biofunctional resources.

BMP-2-immobilized PCL 3D printing scaffold with a leaf-stacked structure as a physically and biologically activated bone graft.

Although three-dimensional (3D) printing techniques are used to mimic macro- and micro-structures as well as multi-structural human tissues in tissue engineering, efficient target tissue regeneration requires bioactive 3D printing scaffolds. In this study, we developed a bone morphogenetic protein-2 (BMP-2)-immobilized polycaprolactone (PCL) 3D printing scaffold with leaf-stacked structure (LSS) (3D-PLSS-BMP) as a bioactive patient-tailored bone graft. The unique LSS was introduced on the strand surface of the scaffold via heating/cooling in tetraglycol without significant deterioration in physical properties. The BMP-2 adsorbed on3D-PLSS-BMPwas continuously released from LSS over a period of 32 d. The LSS can be a microtopographical cue for improved focal cell adhesion, proliferation, and osteogenic differentiation.In vitrocell culture andin vivoanimal studies demonstrated the biological (bioactive BMP-2) and physical (microrough structure) mechanisms of3D-PLSS-BMPfor accelerated bone regeneration. Thus, bioactive molecule-immobilized 3D printing scaffold with LSS represents a promising physically and biologically activated bone graft as well as an advanced tool for widespread application in clinical and research fields.

A bioactive microparticle-loaded osteogenically enhanced bioprinted scaffold that permits sustained release of BMP-2.

Extrusion-based bioprinting technology is widely used for tissue regeneration and reconstruction. However, the method that uses only hydrogel as the bioink base material exhibits limited biofunctional properties and needs improvement to achieve the desired tissue regeneration. In this study, we present a three-dimensionally printed bioactive microparticle-loaded scaffold for use in bone regeneration applications. The unique structure of the microparticles provided sustained release of growth factor for > 4 weeks without the use of toxic or harmful substances. Before and after printing, the optimal particle ratio in the bioink for cell viability demonstrated a survival rate of ≥ 85% over 7 days. Notably, osteogenic differentiation and mineralization-mediated by human periosteum-derived cells in scaffolds with bioactive microparticles-increased over a 2-week interval. Here, we present an alternative bioprinting strategy that uses the sustained release of bioactive microparticles to improve biofunctional properties in a manner that is acceptable for clinical bone regeneration applications.

Perirenal adipose tissues as a human elastin source, and optimize the extraction process.

Elastin is very rarely repaired extracellular matrix (ECM) in physiological condition. The commercial human elastin for exogenous medical treatment is very expensive, and has a potential for disease transmission. Animal-origin elastin is relatively low price, but has concerns for xenogeneic immune responses. Considering cost and safety, we focused on the perirenal adipose tissue, donated from healthy young people via donor nephrectomy. Until now, all of the perirenal adipose tissues are discarded as a medical waste after kidney transplantation. In the present study, we applied perirenal adipose tissues as the source of human elastin, and optimized the extraction process to get high purified and quantified elastin. Through pre-processing step, the delipidated and decellularized ECM was prepared. Next, with four different elastin extraction process (acidic solvents, neutral salt, organic solvents or hot alkali method), elastin was extracted, and the concentration of amino acid between each product was compared, and bright-field/electron microscopy, Fourier transform infrared (FT-IR) spectroscopy and cytotoxicity analysis were also performed. As controls, bovine neck ligament-derived and human skin-derived elastin were used. Among the elastin extraction methods, the hot alkali insoluble product showed (1) relatively high positive area of Verhoeff's and low Masson's trichrome stain, (2) 64.24% purity, 159.29 mg/g quantity, and ∼6.37% yield in amino acid analysis, (3) ß-sheet second structure, and (4) thin fiber composed mesh-like sheet structure in SEM image. These values were higher than those of the commercial human skin elastin. When comparing hydrolyzed forms, α-elastin from hot alkali insoluble product showed enhanced cell proliferation and maintained cell properties compared to the κ-elastin. Therefore, we confirmed that the perirenal adipose tissue is an ideal source of human elastin with safety assurance, and the hot alkali process combined with pre-process seems to be the optimal method for elastin extraction with high purity and quantity.

Therapeutic effect of adipose-derived stem cells and BDNF-immobilized PLGA membrane in a rat model of cavernous nerve injury.

INTRODUCTION: Cavernous nerve injury is the main reason for post-prostatectomy erectile dysfunction (ED). Stem cell and neuroprotection therapy are promising therapeutic strategy for ED. AIM: To evaluate the therapeutic efficacy of adipose-derived stem cells (ADSCs) and brain-derived neurotrophic factor (BDNF) immobilized Poly-Lactic-Co-Glycolic (PLGA) membrane on the cavernous nerve in a rat model of post-prostatectomy ED. Methods. Rats were randomly divided into five groups: normal group, bilateral cavernous nerve crush injury (BCNI) group, ADSC (BCNI group with ADSCs on cavernous nerve) group, BDNF-membrane (BCNI group with BDNF/PLGA membrane on cavernous nerve) group, and ADSC/BDNF-membrane (BCNI group with ADSCs covered with BDNF/PLGA membrane on cavernous nerve) group. BDNF was controlled-released for a period of 4 weeks in a BDNF/PLGA porous membrane system. MAIN OUTCOME MEASURES: Four weeks after the operation, erectile function was assessed by detecting the ratio of intra-cavernous pressure (ICP)/mean arterial pressure (MAP). Smooth muscle and collagen content were determined by Masson's trichrome staining. Neuronal nitric oxide synthase (nNOS) expression in the dorsal penile nerve was detected by immunostaining. Phospho-endothelial nitric oxide synthase (eNOS) protein expression and cyclic guanosine monophosphate (cGMP) level of the corpus cavernosum were quantified by Western blotting and cGMP assay, respectively. RESULTS: In the ADSC/BDNF-membrane group, erectile function was significantly elevated, compared with the BCNI and other treated groups. ADSC/BDNF-membrane treatment significantly increased smooth muscle/collagen ratio, nNOS content, phospho-eNOS protein expression, and cGMP level, compared with the BCNI and other treated groups. CONCLUSIONS: ADSCs with BDNF-membrane on the cavernous nerve can improve erectile function in a rat model of post-prostatectomy ED, which may be used as a novel therapy for post-prostatectomy ED.

Hyaluronic acid/mildly crosslinked alginate hydrogel as an injectable tissue adhesion barrier.

Although hyaluronic acid (HA) has been conventionally utilized as a tissue adhesion barrier material, its rapid clearance in the body still remains as a big challenge in the clinical practice. In this study, we prepared a hydrogel of HA embedded in mildly crosslinked alginate (HA/mcALG hydrogel), which is injectable, easily covers injured tissues, and remains stably at the applied site during wound healing (by muco-adhesive HA embedded in the network structure of the mcALG hydrogel). The HA/mcALG hydrogel was highly effective for the prevention of peritoneal tissue adhesion compared to HA and mcALG hydrogels, and did not lead to any abnormal tissue responses during wound healing. The HA/mcALG hydrogel can be a good candidate as an injectable tissue adhesion barrier for clinical applications.

Effects of Location and Volume of Intraosseous Cement on Adjacent Level of Osteoporotic Spine Undergoing Kyphoplasty: Finite Element Analysis.

OBJECTIVE: Kyphoplasty (KP) is a surgery used to reduce pain and increase stability by injecting medical bone cement into broken vertebrae. The purpose of this study was to determine the ideal amount of cement and injection site by analyzing forces with the finite element method. METHODS: We modeled the anatomical structure of the vertebra and injected the cement at T12. By increasing the amount of cement from 1 cc to 22 cc, stress applied to T11 and L1 cortical was calculated. In addition, stress applied to the adjacent KP level was calculated with different injection sites (medial, anterosuperior, posterosuperior, anteroinferior, and posteroinferior). After 5 cc cement was inserted, adjacent end plate stress was analyzed. RESULTS: In this study, break point adjacent bone stress according to the capacity of cement was bimodal. Flexion/extension and lateral bending conditions showed similar break points (11.5-11.7 cc and 18.5-18.6 cc, respectively). When cement injection was changed, front under and back under had the highest stress values among various parts, whereas the center position showed the lowest stress value. CONCLUSIONS: With increasing amount of bone cement, stress on the upper and lower end plates of the cemented segment increased significantly. Thus, increasing cement amount to be more than 11.5 cc has a potential risk of adjacent fracture. Centrally injected bone cement can lower the risk of adjacent fracture after percutaneous KP.

Bioactive Porous Particles as Biological and Physical Stimuli for Bone Regeneration.

Even though bony defects can be recovered to their original condition with full functionality, critical-sized bone injuries continue to be a challenge in clinical fields due to deficiencies in the scaffolding matrix and growth factors at the injury region. In this study, we prepared bone morphogenetic protein-2 (BMP-2)-loaded porous particles as a bioactive bone graft for accelerated bone regeneration. The porous particles with unique leaf-stacked morphology (LSS particles) were fabricated by a simple cooling procedure of hot polycaprolactone (PCL) solution. The unique leaf-stacked structure in the LSS particles provided a large surface area and complex release path for the sufficient immobilization of BMP-2 and sustained release of BMP-2 for 26 days. The LSS was also recognized as a topographical cue for cell adhesion and differentiation. In in vitro cell culture and in vivo animal study using a canine mandible defect model, BMP-2-immobilized LSS particles provided a favorable environment for osteogenic differentiation of stem cells and bone regeneration. In vitro study suggests a dual stimulus of bone mineral-like (leaf-stacked) structure (a physical cue) and continuously supplied BMP-2 (a biological cue) to be the cause of this improved healing outcome. Thus, LSS particles containing BMP-2 can be a promising bioactive grafting material for effective new bone formation.

3D Spheroid Formation Using BMP-Loaded Microparticles Enhances Odontoblastic Differentiation of Human Dental Pulp Stem Cells.

Human dental pulp stem cells (hDPSCs) are the primary cells responsible for dentin regeneration. Typically, in order to allow for odontoblastic differentiation, hDPSCs are cultured over weeks with differentiation-inducing factors in a typical monolayered culture. However, monolayered cultures have significant drawbacks including inconsistent differentiation efficiency, require a higher BMP concentration than should be necessary, and require periodic treatment with BMPs for weeks to see results. To solve these problems, we developed a 3D-cell spheroid culture system for odontoblastic differentiation using microparticles with leaf-stacked structure (LSS), which allow for the sustained release of BMPs and adequate supply of oxygen in cell spheroids. BMPs were continuously released and maintained an effective concentration over 37 days. hDPSCs in the spheroid maintained their viability for 5 weeks, and the odontoblastic differentiation efficiency was increased significantly compared to monolayered cells. Finally, dentin-related features were detected in the spheroids containing BMPs-loaded microparticles after 5 weeks, suggesting that these hDPSC-LSS spheroids might be useful for dentin tissue regeneration.

The effects of VEGF-centered biomimetic delivery of growth factors on bone regeneration.

It is accepted that biomimetic supply of signaling molecules during bone regeneration can provide an appropriate environment for accelerated new bone formation. In this study, we developed a growth factor delivery system based on porous particles and a thermosensitive hydrogel that allowed fast, continuous, and delayed/continuous release of growth factors to mimic their biological production during bone regeneration. It was observed that the Continuous group (continuous release of growth factors) provides a better environment for the osteogenic differentiation of hPDCs than the Biomimetic group (biomimetic release of growth factors), and thus is anticipated to promote bone regeneration. However, contrary to expectation, the Biomimetic group promoted significant new bone formation compared to the Continuous group. From the systematic cell culture experiments, the initial supply of VEGF was considered to have more favorable effects on the osteoclastogenesis than osteogenesis, which may hinder bone regeneration. Our results indicated that the continuous supply of VEGF (in particular, at early stage) from VEGF-loaded biomaterial might not be conducive to new bone formation. Therefore, we suggest that a biomimetic supply of growth factors is a more pivotal parameter for sufficient tissue regeneration. Its use as a molecular delivery system may also serve as a useful tool for the investigation of biological processes and molecules during tissue regeneration processes.