Enhanced intra-articular therapy for rheumatoid arthritis using click-crosslinked hyaluronic acid hydrogels loaded with toll-like receptor antagonizing peptides.

Rheumatoid arthritis (RA) is a chronic inflammatory disorder that results in the deterioration of joint cartilage and bone. Toll-like receptor 4 (TLR4) has been pinpointed as a key factor in RA-related inflammation. While Toll-like receptor antagonizing peptide 2 (TAP2) holds potential as an anti-inflammatory agent, its in vivo degradation rate hinders its efficacy. We engineered depots of TAP2 encapsulated in click-crosslinked hyaluronic acid (TAP2+Cx-HA) for intra-articular administration, aiming to enhance the effectiveness of TAP2 as an anti-inflammatory agent within the joint cavity. Our data demonstrated that FI-TAP2+Cx-HA achieves a longer retention time in the joint cavity compared to FI-TAP2 alone. Mechanistically, we found that TAP2 interacts with TLR4 on the cell membranes of inflammatory cells, thereby inhibiting the nuclear translocation of NF-κB and maintaining it in an inactive cytoplasmic state. In a rat model of RA, the TAP2+Cx-HA formulation effectively downregulated the inflammatory cytokines TNF-α and IL-6, while upregulating the anti-inflammatory cytokine IL-10 and the therapeutic protein 14-3-3ζ. This led to a more rapid restoration of cartilage thickness, increased deposition of glycosaminoglycans, and new bone tissue formation in the regenerated cartilage, in comparison to a single TAP2 treatment after a six-week period. Our results suggest that TAP2+Cx-HA could serve as a potent intra-articular treatment for RA, offering both symptomatic relief and promoting cartilage regeneration. This innovative delivery system holds significant promise for improving the management of RA and other inflammatory joint conditions. STATEMENT OF SIGNIFICANCE: In this study, we developed a therapy by creating toll-like receptor 4 (TLR4)-antagonizing peptide (TAP2)-loaded click-crosslinked hyaluronic acid (TAP2+Cx-HA) depots for direct intra-articular injection. Our study demonstrates that FI-TAP2+Cx-HA exhibits a more than threefold longer lifetime in the joint cavity compared to FI-TAP2 alone. Furthermore, we found that TAP2 binds to TLR4 and masks the nuclear localization signals of NF-κB, leading to its sequestration in an inactive state in the cytoplasm. In a rat model of RA, TAP2+Cx-HA effectively suppresses inflammatory molecules, specifically TNF-α and IL-6, while upregulating the anti-inflammatory cytokine IL-10 and the therapeutic protein 14-3-3ζ. This resulted in faster regeneration of cartilage thickness, increased glycosaminoglycan deposits in the regenerated cartilage, and a twofold increase in new bone tissue formation compared to a single TAP2 treatment.

Preparation and evaluation of injectable microsphere formulation for longer sustained release of donepezil.

In this study, donepezil-loaded PLGA and PLA microspheres (Dp-PLGA-M/Dp-PLA-M) and Dp-PLA-M wrapped in a polyethylene glycol-b-polycaprolactone (PC) hydrogel (Dp-PLA-M/PC) were prepared to reduce the dosing frequency of injections to treat Alzheimer's disease patients. Dp-PLGA-M and Dp-PLA-M with a uniform particle size distribution were repeatably fabricated in nearly quantitative yield and with high encapsulated Dp yields using an ultrasonic atomizer. The injectability and in vitro and in vivo Dp release, biodegradation, and inflammatory response elicited by the Dp-PLGA-M, Dp-PLA-M, and Dp-PLA-M/PC formulations were then compared. All injectable formulations showed good injectability with ease of injection, even flow, and no clogging using a syringe needle under 21-G. The injections required a force of <1 N. According to the biodegradation rate of micro-CT, GPC and NMR analyses, the biodegradation of Dp-PLA-M was slower than that of Dp-PLGA-M, and the biodegradation rate of Dp-PLA-M/PC was also slower. In the Dp release experiment, Dp-PLA-M sustained Dp for longer compared with Dp-PLGA-M. Dp-PLA-M/PC exhibited a longer sustained release pattern of two months. In vivo bioavailability of Dp-PLA-M/PC was almost 1.4 times higher than that of Dp-PLA-M and 1.9 times higher than that of Dp-PLGA-M. The variations in the Dp release patterns of Dp-PLGA-M and Dp-PLA-M were explained by differences in the degradation rates of PLGA and PLA. The sustained release of Dp by Dp-PLA-M/PC was attributed to the fact that the PC hydrogel served as a wrapping matrix for Dp-PLA-M, which could slow down the biodegradation of PLA-M, thus delaying the release of Dp from Dp-PLA-M. Dp-PLGA-M induced a higher inflammatory response compared to Dp-PLA-M/PC, suggesting that the rapid degradation of PLGA triggered a strong inflammatory response. In conclusion, Dp-PLA-M/PC is a promising injectable Dp formulation that could be used to reduce the dosing frequency of Dp injections.

An injectable cationic hydrogel electrostatically interacted with BMP2 to enhance in vivo osteogenic differentiation of human turbinate mesenchymal stem cells.

We have designed and characterized an injectable, electrostatically bonded, in situ-forming hydrogel system consisting of a cationic polyelectrolyte [(methoxy)polyethylene glycol-b-(poly(ε-caprolactone)-ran-poly(L-lactic acid)] (MP) copolymer derivatized with an amine group (MP-NH2) and anionic BMP2. To the best of our knowledge, there have been hardly any studies that have investigated electrostatically bonded, in situ-forming hydrogel systems consisting of MP-NH2 and BMP2, with respect to how they promote in vivo osteogenic differentiation of human turbinate mesenchymal stem cells (hTMSCs). Injectable formulations almost immediately formed an electrostatically loaded hydrogel depot containing BMP2, upon injection into mice. The hydrogel features and stability of BMP2 inside the hydrogel were significantly affected by the electrostatic attraction between BMP2 and MP-NH2. Additionally, the time BMP2 spent inside the hydrogel depot was prolonged in vivo, as evidenced by in vivo near-infrared fluorescence imaging. Biocompatibility was demonstrated by the fact that hTMSCs survived in vivo, even after 8 weeks and even though relatively few macrophages were in the hydrogel depot. The osteogenic capacity of the electrostatically loaded hydrogel implants containing BMP2 was higher than that of a hydrogel that was simply loaded with BMP2, as evidenced by Alizarin Red S, von Kossa, and hematoxylin and eosin staining as well as osteonectin, osteopontin, osteocalcin, and type 1α collagen mRNA expression. The results confirmed that our injectable, in situ-forming hydrogel system, electrostatically loaded with BMP2, can enhance in vivo osteogenic differentiation of hTMSCs.

Bone regeneration by means of a three-dimensional printed scaffold in a rat cranial defect.

Recently, computer-designed three-dimensional (3D) printing techniques have emerged as an active research area with almost unlimited possibilities. In this study, we used a computer-designed 3D scaffold to drive new bone formation in a bone defect. Poly-L-lactide (PLLA) and bioactive ß-tricalcium phosphate (TCP) were simply mixed to prepare ink. PLLA + TCP showed good printability from the micronozzle and solidification within few seconds, indicating that it was indeed printable ink for layer-by-layer printing. In the images, TCP on the surface of (and/or inside) PLLA in the printed PLLA + TCP scaffold looked dispersed. MG-63 cells (human osteoblastoma) adhered to and proliferated well on the printed PLLA + TCP scaffold. To assess new bone formation in vivo, the printed PLLA + TCP scaffold was implanted into a full-thickness cranial bone defect in rats. The new bone formation was monitored by microcomputed tomography and histological analysis of the in vivo PLLA + TCP scaffold with or without MG-63 cells. The bone defect was gradually spontaneously replaced with new bone tissues when we used both bioactive TCP and MG-63 cells in the PLLA scaffold. Bone formation driven by the PLLA + TCP30 scaffold with MG-63 cells was significantly greater than that in other experimental groups. Furthermore, the PLLA + TCP scaffold gradually degraded and matched well the extent of the gradual new bone formation on microcomputed tomography. In conclusion, the printed PLLA + TCP scaffold effectively supports new bone formation in a cranial bone defect.

Preparation of Biodegradable and Elastic Poly(ε-caprolactone-co-lactide) Copolymers and Evaluation as a Localized and Sustained Drug Delivery Carrier.

To develop a biodegradable polymer possessing elasticity and flexibility, we synthesized MPEG-b-(PCL-co-PLA) copolymers (PCxLyA), which display specific rates of flexibility and elasticity. We synthesize the PCxLyA copolymers by ring-opening polymerization of ε-caprolactone and l-lactide. PCxLyA copolymers of various compositions were synthesized with 500,000 molecular weight. The PCxLyA copolymers mechanical properties were dependent on the mole ratio of the ε-caprolactone and l-lactide components. Cyclic tensile tests were carried out to investigate the resistance to creep of PCxLyA specimens after up to 20 deformation cycles to 50% elongation. After in vivo implantation, the PCxLyA implants exhibited biocompatibility, and gradually biodegraded over an eight-week experimental period. Immunohistochemical characterization showed that the PCxLyA implants provoked in vivo inflammation, which gradually decreased over time. The copolymer was used as a drug carrier for locally implantable drugs, the hydrophobic drug dexamethasone (Dex), and the water-soluble drug dexamethasone 21-phosphate disodium salt (Dex(p)). We monitored drug-loaded PCxLyA films for in vitro and in vivo drug release over 40 days and observed real-time sustained release of near-infrared (NIR) fluorescence over an extended period from hydrophobic IR-780- and hydrophilic IR-783-loaded PCxLyA implanted in live animals. Finally, we confirmed that PCxLyA films are usable as biodegradable, elastic drug carriers.

Evaluation of small intestine submucosa and poly(caprolactone-co-lactide) conduits for peripheral nerve regeneration.

The present study employed nerve guidance conduits (NGCs) only, which were made of small intestine submucosa (SIS) and poly(caprolactone-co-lactide) (PCLA) to promote nerve regeneration in a peripheral nerve injury (PNI) model with nerve defects of 15 mm. The SIS- and PCLA-NGCs were easily prepared by rolling of a SIS sheet and a bioplotter using PCLA, respectively. The prepared SIS- and PCLA-NGCs fulfilled the general requirement for use as artificial peripheral NGCs such as easy fabrication, reproducibility for mass production, suturability, sterilizability, wettability, and proper mechanical properties to resist collapsing when applied to in vivo implantation. The SIS- and PCLA-NGCs appeared to be well integrated into the host sciatic nerve without causing dislocations and serious inflammation. All NGCs stably maintained their NGC shape for 8 weeks without collapsing, which matched well with the nerve regeneration rate. Staining of the NGCs in the longitudinal direction showed that the regenerated nerves grew successfully from the SIS- and PCLA-NGCs through the sciatic nerve-injured gap and connected from the proximal to distal direction along the NGC axis. SIS-NGCs exhibited a higher nerve regeneration rate than PCLA-NGCs. Collectively, our results indicate that SIS- and PCLA-NGCs induced nerve regeneration in a PNI model, a finding that has significant implications in the future with regard to the feasibility of clinical nerve regeneration with SIS- and PCLA-NGCs prepared through an easy fabrication method using promising biomaterials.

Perspectives on tissue-engineered nerve regeneration for the treatment of spinal cord injury.

Over the past few decades, substantial progress has been made to safely improve nerve function in spinal cord injury (SCI) patients through the regeneration of injured nerve tissue. This perspective focuses on an extensive overview of SCI research as well as tissue-engineered nerve regeneration for the treatment of SCI.

Cellular behavior of human adipose-derived stem cells on wettable gradient polyethylene surfaces.

Appropriate surface wettability and roughness of biomaterials is an important factor in cell attachment and proliferation. In this study, we investigated the correlation between surface wettability and roughness, and biological response in human adipose-derived stem cells (hADSCs). We prepared wettable and rough gradient polyethylene (PE) surfaces by increasing the power of a radio frequency corona discharge apparatus with knife-type electrodes over a moving sample bed. The PE changed gradually from hydrophobic and smooth surfaces to hydrophilic (water contact angle, 90° to ~ 50°) and rough (80 to ~120 nm) surfaces as the power increased. We found that hADSCs adhered better to highly hydrophilic and rough surfaces and showed broadly stretched morphology compared with that on hydrophobic and smooth surfaces. The proliferation of hADSCs on hydrophilic and rough surfaces was also higher than that on hydrophobic and smooth surfaces. Furthermore, integrin beta 1 gene expression, an indicator of attachment, and heat shock protein 70 gene expression were high on hydrophobic and smooth surfaces. These results indicate that the cellular behavior of hADSCs on gradient surface depends on surface properties, wettability and roughness.

In Vivo Osteogenic Differentiation of Human Embryoid Bodies in an Injectable in Situ-Forming Hydrogel.

In this study, we examined the in vivo osteogenic differentiation of human embryoid bodies (hEBs) by using an injectable in situ-forming hydrogel. A solution containing MPEG-b-(polycaprolactone-ran-polylactide) (MCL) and hEBs was easily prepared at room temperature. The MCL solution with hEBs and osteogenic factors was injected into nude mice and developed into in situ-forming hydrogels at the injection sites; these hydrogels maintained their shape even after 12 weeks in vivo, thereby indicating that the in situ-forming MCL hydrogel was a suitable scaffold for hEBs. The in vivo osteogenic differentiation was observed only in the in situ gel-forming MCL hydrogel in the presence of hEBs and osteogenic factors. In conclusion, this preliminary study suggests that hEBs and osteogenic factors embedded in an in situ-forming MCL hydrogel may provide numerous benefits as a noninvasive alternative for allogeneic tissue engineering applications.

Tissue engineered regeneration of completely transected spinal cord using human mesenchymal stem cells.

The present study employed a combinatorial strategy using poly(D,L-lactide-co-glycolide) (PLGA) scaffolds seeded with human mesenchymal stem cells (hMSCs) to promote cell survival, differentiation, and neurological function in a completely transected spinal cord injury (SCI) model. The SCI model was prepared by complete removal of a 2-mm length of spinal cord in the eighth-to-ninth spinal vertebra, a procedure that resulted in bilateral hindlimb paralysis. PLGA scaffolds 2 mm in length without hMSCs (control) or with different numbers of hMSCs (1 × 10(5), 2 × 10(4), and 4 × 10(3)) were fitted into the completely transected spinal cord. Rats implanted with hMSCs received Basso-Beattie-Bresnahan scores for hindlimb locomotion of about 5, compared with ~2 for animals in the control group. The amplitude of motor-evoked potentials (MEPs) averaged 200-300 µV in all hMSC-implanted SCR model rats. In contrast, the amplitude of MEPs in control group animals averaged 135 µV at 4 weeks and then declined to 100 µV at 8 weeks. These results demonstrate functional recovery in a completely transected SCI model under conditions that exclude self-recovery. hMSCs were detected at the implanted site 4 and 8 weeks after transplantation, indicating in vivo survival of implanted hMSCs. Immunohistochemical staining revealed differentiation of implanted hMSCs into nerve cells, and immunostained images showed clear evidence for axonal regeneration only in hMSC-seeded PLGA scaffolds. Collectively, our results indicate that hMSC-seeded PLGA scaffolds induced nerve regeneration in a completely transected SCI model, a finding that should have significant implications for the feasibility of therapeutic and clinical hMSC-delivery using three-dimensional scaffolds, especially in the context of complete spinal cord transection.

In vivo biofunctionality comparison of different topographic PLLA scaffolds.

In this work, the in vivo biodegradation of, biocompatibility of, and host response to various topographic scaffolds were investigated. Randomly oriented fibrous poly(L-lactide) (PLLA) nanofibers were fabricated using the electrospinning technique. A PLLA scaffold was obtained by salt leaching. Both the electrospun PLLA nanofibers and the salt-leaching PLLA scaffolds formed three-dimensional pore structures. Cytotoxicity studies, in which rat muscle-derived stem cells (rMDSCs) were grown on electrospun PLLA nanofibers or the salt-leaching PLLA scaffolds, revealed that the rMDSCs cell count on the PLLA nanofibers was slightly higher than that on the salt-leaching PLLA scaffolds. An in vivo study was carried out by implanting the scaffolds subcutaneously into rats to test the biodegradation, biocompatibility, and host response at regular intervals over 0-4 weeks. The degradation of the PLLA nanofibers 1, 2, and 4 weeks after initial implantation was more extensive than that observed for the salt-leaching PLLA scaffolds. PLLA nanofibers seeded the growth of larger fibrous tissue masses due to in vivo cellular infiltration into the randomly oriented fibrillar structures of the PLLA nanofibers. In addition, the inflammatory cell accumulation in PLLA nanofibers was lower than that in the salt-leaching PLLA scaffolds. These results indicate that the electrospun PLLA nanofibers may serve as a good scaffold to elicit fibrous cellular infiltration, to minimize host response, and to enhance tissue-scaffold integration.

In vivo release of bovine serum albumin from an injectable small intestinal submucosa gel.

We aimed to develop a delivery system capable of maintaining a sustained release of protein drugs at specific sites using potentially biocompatible biomaterials. Here, we used bovine serum albumin (BSA) as a test protein to explore the potential utility of an injectable small intestine submucosa (SIS) as a depot for protein drugs. The prepared SIS powder was dispersed in PBS. The SIS suspension easily entrapped BSA in pharmaceutical formulations at room temperature. When this was suspension subcutaneously injected into rats, it gelled, forming an interconnecting three-dimensional network SIS structure to allow BSA to penetrate through it. The amount of BSA-FITC released from the SIS gel was determined in rat plasma and monitored by real-time in vivo molecular imaging. The data indicated the sustained release of BSA-FITC for 30 days in vivo. In addition, SIS gel provoked little inflammatory response. Collectively, our results show that the SIS gel described here could serve as a minimally invasive therapeutics depot with numerous benefits compared to other injectable biomaterials.

Regeneration of completely transected spinal cord using scaffold of poly(D,L-lactide-co-glycolide)/small intestinal submucosa seeded with rat bone marrow stem cells.

Using a complete spinal cord transection model, the present study employed a combinatorial strategy comprising rat bone marrow stem cells (rBMSCs) and polymer scaffolds to regenerate neurological function after spinal cord injury (SCI) of different lengths. SCI models with completely transected lesions were prepared by surgical removal of 1 mm (SC1) or 3 mm (SC3) lengths of spinal cord in the eighth-to-ninth spinal vertebrae, a procedure that resulted in bilateral hindlimb paralysis. A cylindrical poly(D,L-lactide-co-glycolide)/small intestinal submucosa scaffold 1 or 3 mm in length with or without rBMSCs was fitted into the completely transected lesion. Rats in SC1 and SC3 groups implanted with rBMSC-containing scaffolds received Basso-Beattie-Bresnahan scores for hindlimb locomotion of 15 and 8, respectively, compared with ∼3 for control rats in SC1-C and SC3-C groups implanted with scaffolds lacking rBMSCs. The amplitude of motor-evoked potentials recorded in the hindlimb area of the sensorimotor cortex after stimulation of the injured spinal cord averaged ∼100 µV in SC1-C and 10-50 µV in SC3-C groups at 4 weeks, and then declined to nearly zero at 8 weeks. In contrast, the amplitude of motor-evoked potentials increased from ∼300 to 350 µV between 4 and 8 weeks in SC1 rats and from ∼200 to ∼250 µV in SC3 rats. These results demonstrate functional recovery in rBMSC-transplanted rats, especially those with smaller defects. Immunohistochemically stained sections of the injury site showed clear evidence for axonal regeneration only in rBMSC-transplanted SC1 and SC3 models. In addition, rBMSCs were detected at the implanted site 4 and 8 weeks after transplantation, indicating cell survival in SCI. Collectively, our results indicate that therapeutic rBMSCs in a poly(D,L-lactide-co-glycolide)/small intestinal submucosa scaffold induced nerve regeneration in a complete spinal cord transection model and showed that functional recovery further depended on defect length.

In vivo efficacy of paclitaxel-loaded injectable in situ-forming gel against subcutaneous tumor growth.

Injectable in situ-forming gels have received considerable attention as localized drug delivery systems. Here, we examined a poly(ethylene glycol)-b-polycaprolactone (MPEG-PCL) diblock copolymer gel as an injectable drug depot for paclitaxel (Ptx). The copolymer solution remained liquid at room temperature and rapidly gelled in vivo at body temperature. In vitro experiments showed that Ptx was released from MPEG-PCL copolymer gels over the course of more than 14 days. Experiments employing intratumoral injection of saline (control), gel-only, Taxol, or Ptx-loaded gel into mice bearing B16F10 tumor xenografts showed that Ptx-loaded gel inhibited the growth of B16F10 tumors more effectively than did saline or gel alone. Further, intratumoral injection of Ptx-loaded gel was more efficacious in inhibiting the growth of B16F10 tumor over 10 days than was injection of Taxol. A histological analysis demonstrated an increase in necrotic tissue in tumors treated with Ptx-loaded gel. In conclusion, our data show that intratumoral injection of Ptx-loaded MPEG-PCL diblock copolymer yielded an in situ-forming gel that exhibited controlled Ptx release profile, and that was effective in treating localized solid tumors.

Small intestine submucosa sponge for in vivo support of tissue-engineered bone formation in the presence of rat bone marrow stem cells.

The aim of the current study was to visualize new bone formed in vivo on a small intestine submucosa (SIS) sponge used as a tissue-engineered scaffold for the repair of damaged bone. The SIS sponge provided a three-dimensional pore structure, and supported good attachment and viability of rat bone marrow stem cells (rBMSCs). To examine bone regeneration, we prepared full-thickness bilateral bone defects in the rat crania, and then treated the defects with an implanted SIS sponge or PGA mesh without or with rBMSCs, or left the defects untreated. Bone defects were evaluated by micro-CT and histologically after 2 and 4 weeks. Micro-CT demonstrated a trend toward a decrease in bone void in both the SIS sponge and SIS sponge/rBMSCs groups compared to the control and PGA mesh groups. At 4 weeks, bone formation in defects containing SIS sponge/rBMSCs was significantly greater than in all other groups. A histological analysis after 2 and 4 weeks of implantation showed localized collagen and osteocalcin deposition on SIS sponges and SIS sponges with rBMSCs. These in vivo results indicate that the SIS sponge, implanted at bone-removal defects, facilitated bone regeneration.

Insulin-loaded microcapsules for in vivo delivery.

Microencapsulation of insulin has been difficult, due to the high sensitivity of insulin to the harsh conditions that can occur during the microencapsulation process. We have developed a method of preparing insulin-loaded microcapsules by using a monoaxial ultrasonic atomizer to form microdroplets of insulin in aqueous solution surrounded by poly(lactic-co-glycolic acid) (PLGA) solution. Administration of these insulin-loaded microcapsules to type 1 diabetic rats maintained plasma insulin concentrations for 30 days, due to the sustained insulin release properties of the microcapsules. In contrast, plasma insulin concentrations after subcutaneous injection of insulin solution reached near zero levels within 2 days. Insulin solution showed only an immediate pharmacological effect, with no reduction of glycemia after 3 days, whereas insulin-loaded microcapsules maintained blood glucose levels at 100-200 mg/dL for 55 days. Molecular imaging using fluorescein isothiocyanate (FITC)-insulin-loaded microcapsules showed in vivo sustained release of the FITC-insulin in microcapsules. Using insulin-loaded microcapsules, we observed inflammation only immediately after injection, indicating that the rats adapted to long-term insulin release. In conclusion, insulin-loaded microcapsules may reduce nonrepetitive insulin administration and show sustained pharmacological performance.

Induction of neurogenesis in rat bone marrow mesenchymal stem cells using purine structure-based compounds.

Rat bone marrow stem cells (rBMSCs) in the presence of chemical molecules were differentiated into neurons.

Electrostatic crosslinked in situ-forming in vivo scaffold for rat bone marrow mesenchymal stem cells.

We herein formulated and characterized an in situ-forming gel consisting of sodium carboxymethylcellulose (CMC) and poly(ethyleneimine) (PEI) and examined its use as an in vivo scaffold for rat bone marrow stem cells (rBMSCs). The changes of zeta potential, size, and viscosity of CMC solutions with 0 to 30 wt% PEI confirmed the electrostatic interaction and temperature-dependence between anionic CMC and cationic PEI. The CMC/PEI solution produced an electrostatically crosslinked gel with a three-dimensional network structure. The CMC solution containing 10 wt% PEI transformed to a gel at temperatures greater than 35 degrees C and was chosen for subcutaneous injection into rats. The CMC/PEI (90/10) gel with pore structure acted as a suitable biocompatible substrate for the in vitro and in vivo attachment and proliferation of rBMSCs. As the CMC/PEI (90/10) solution with and without rBMSCs was injected into Fisher rats, it became a gel in the subcutaneous dorsum of the rats and maintained its shape even after 28 days in vivo. The injected rBMSCs survived in the CMC gel for 28 days. Injection of CMC/PEI gel alone induced macrophage accumulation in the host tissue and at the edge of the gel, whereas injection of CMC/PEI gel containing rBMSCs was associated with less macrophage accumulation, indicating immunosuppression by the transplanted rBMSCs. Our results collectively show that CMC/PEI gel could serve as an in situ-forming gel scaffold for entrapped rBMSCs in vivo.

In vivo osteogenic differentiation of human adipose-derived stem cells in an injectable in situ-forming gel scaffold.

The sol-to-gel transition occurring at around body temperature makes the MPEG-PCL diblock copolymer an ideal candidate material for use as an injectable in situ-forming gel containing human adipose tissue-derived stem cells (hADSCs). The sol can be prepared at room temperature, and the gel forms at body temperature. Solutions of the copolymer containing hADSCs and osteogenic factors injected into rats formed gel scaffolds at the injection sites. The gels thus formed showed the interconnective pore structure required to support growth, proliferation, and differentiation of hADSCs. Bromodeoxyuridine-labeled hADSCs were confirmed to be present in gels formed in vivo. Bone formation was observed only in gel implants containing both hADSCs and osteogenic factors. Subcutaneous implantation of the in situ-forming gel scaffold demonstrated that hADSCs embedded in the gel stimulated much lower host tissue responses than did the gel alone, probably because of the unique immunomodulatory properties of hADSCs. In conclusion, our data on hADSCs embedded in an in situ gel scaffold suggest that this formulation may provide numerous benefits as a noninvasive alternative for tissue-engineered bone formation.

Injectable CMC/PEI gel as an in vivo scaffold for demineralized bone matrix.

A number of materials have been considered as sources of grafts to repair bone defects. Here, we examined the possibility of creating in situ-forming gels from sodium carboxymethylcellulose (CMC) and poly(ethyleneimine) (PEI) for use as an in vivo carrier of demineralized bone matrix (DBM). The interaction between anionic CMC and cationic PEI was examined by evaluating phase transition behavior and viscosity of CMC solutions containing 0-30 wt% PEI. CMC solutions containing 10 wt% PEI exhibited a sol-to-gel phase transition at temperatures greater than 35 degrees C. The phase transition is caused by electrostatic crosslinking of the CMC/PEI solution to form a gel with a three-dimensional network structure. In situ-formed gel implants were successfully fabricated in vivo by simple subcutaneous injection of the CMC/PEI (90/10) solution (with and without DBM) into Fisher rats. The resulting in situ-formed implant maintained its shape for 28 days in vitro and in vivo. Our results show that in situ-forming CMC/PEI gels can serve as a DBM carrier that can be delivered with a minimally invasive procedure.