Comparison of Scaffolds Fabricated via 3D Printing and Salt Leaching: In Vivo Imaging, Biodegradation, and Inflammation.

In this work, we prepared fluorescently labeled poly(ε-caprolactone-ran-lactic acid) (PCLA-F) as a biomaterial to fabricate three-dimensional (3D) scaffolds via salt leaching and 3D printing. The salt-leached PCLA-F scaffold was fabricated using NaCl and methylene chloride, and it had an irregular, interconnected 3D structure. The printed PCLA-F scaffold was fabricated using a fused deposition modeling printer, and it had a layered, orthogonally oriented 3D structure. The printed scaffold fabrication method was clearly more efficient than the salt leaching method in terms of productivity and repeatability. In the in vivo fluorescence imaging of mice and gel permeation chromatography of scaffolds removed from rats, the salt-leached PCLA scaffolds showed slightly faster degradation than the printed PCLA scaffolds. In the inflammation reaction, the printed PCLA scaffolds induced a slightly stronger inflammation reaction due to the slower biodegradation. Collectively, we can conclude that in vivo biodegradability and inflammation of scaffolds were affected by the scaffold fabrication method.

Preparation of a cross-linked cartilage acellular matrix-poly (caprolactone-ran-lactide-ran-glycolide) film and testing its feasibility as an anti-adhesive film.

To protect unwanted tissue adhesions occurring after surgeries, we aimed to fabricate an anti-adhesive film using cartilage acellular matrix (CAM) with anti-vascular inhibition activity. Additionally, to fabricate anti-adhesive films with tunable swelling, mechanical, and biodegradation properties, a biodegradable polyester (PEP) with N-hydroxysuccinimide (NHS) in the chain end position was synthesized as a cross-linker. CAM/PEP (CP) films were prepared with various CAM: PEP ratios in the wide size with repeatable reproducibility, and then, cross-linked CP (Cx-CP) were obtained by the interpenetrating cross-linking reaction between the amine group on CAM and the NHS group on PEP cross-linkers under thermal treatment. The biodegradation, wettability, swelling, and mechanical properties of the prepared anti-adhesive Cx-CP films were controlled by varying the CAM:PEP ratio. The degradation half-life, contact angle, elastic moduli and toughness of Cx-CP films increased according to the increasing PEP content. Additionally, Cx-CP films significantly inhibits the attachment and proliferation of HUVECs. Cx-CP film prepared by varying the CAM:PEP ratio can be tailored to meet individual requirements for in vivo injured tissues. In animal experiments, anti-adhesive Cx-CP films implanted between the peritoneal wall and the cecum significantly suppressed tissue adhesion between them. Additionally, good adhesion effect observed at anti-adhesive film maintained for proper time period at injured tissues. Taken together, in this work, we successfully achieved strategy for the development of anti-adhesive barrier with tunable swelling, mechanical, and biodegradation properties.

An injectable click-crosslinked hyaluronic acid hydrogel modified with a BMP-2 mimetic peptide as a bone tissue engineering scaffold.

An injectable, click-crosslinking (Cx) hyaluronic acid (HA) hydrogel scaffold modified with a bone morphogenetic protein-2 (BMP-2) mimetic peptide (BP) was prepared for bone tissue engineering applications. The injectable click-crosslinking HA formulation was prepared from HA-tetrazine (HA-Tet) and HA-cyclooctene (HA-TCO). The Cx-HA hydrogel scaffold was prepared simply by mixing HA-Tet and HA-TCO. The Cx-HA hydrogel scaffold was stable for a longer period than HA both in vitro and in vivo, which was verified via in-vivo fluorescence imaging in real time. BP acted as an osteogenic differentiation factor for human dental pulp stem cells (hDPSCs). After its formation in vivo, the Cx-HA scaffold provided a fine environment for the hDPSCs, and the biocompatibility of the hydrogel scaffold with tissue was good. Like traditional BMP-2, BP induced the osteogenic differentiation of hDPSCs in vitro. The physical properties and injectability of the chemically loaded BP for the Cx-HA hydrogel (Cx-HA-BP) were nearly identical to those of the physically loaded BP hydrogels and the Cx-HA-BP formulation quickly formed a hydrogel scaffold in vivo. The chemically loaded hydrogel scaffold retained the BP for over a month. The Cx-HA-BP hydrogel was better at inducing the osteogenic differentiation of loaded hDPSCs, because it prolonged the availability of BP. In summary, we successfully developed an injectable, click-crosslinking Cx-HA hydrogel scaffold to prolong the availability of BP for efficient bone tissue engineering.

Injectable In Situ-Forming Hydrogels for Protein and Peptide Delivery.

Injectable in situ-forming hydrogels have been used clinically in diverse biomedical applications. These hydrogels have distinct advantages such as easy management and minimal invasiveness. The hydrogels are aqueous formulations, and a simple injection at the target site replaces a traditional surgical procedure. Here, we review injectable in situ-forming hydrogels that are formulated by physical and chemical methods to deliver proteins and peptides. Prospects for using in situ-forming hydrogels for several specific applications are also discussed.

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.

Preparation of a Cross-Linked Cartilage Acellular-Matrix Film and Its In Vivo Evaluation as an Antiadhesive Barrier.

In this paper, a cartilage acellular-matrix (CAM) is chosen as a biomaterial for an effective antiadhesive barrier to apply between injured tissue and healthy tissues or organs. CAM is cross-linked using glutaraldehyde to create a cross-linked CAM (Cx-CAM) film. Cx-CAM has higher elastic modulus and toughness and more hydrophobic surface properties than CAM before cross-linking. Small intestinal submucosa (SIS), cross-linked SIS (Cx-SIS) as a negative control, and Seprafilm as a positive control are used in an experiment as adhesion barriers. Human umbilical vein endothelial cells (HUVECs) on SIS, Cx-SIS, or in a culture plate get attached and effectively proliferate for 7 days, but Cx-CAM and Seprafilm allow for little or no attachment and proliferation of HUVECs, thus manifesting antiadhesive and antiproliferative effects. In animals with surgical damage to the peritoneal wall and cecum, Cx-CAM and Seprafilm afford little adhesion and negligible inflammation after seven days, as confirmed by hematoxylin and eosin staining and macrophage staining, in contrast to an untreated-injury model, SIS, or Cx-SIS film. Cx-CAM significantly suppresses the formation of blood vessels between the peritoneal wall and cecum, as confirmed by CD31 staining. Overall, the newly designed Cx-CAM film works well as an antiadhesion barrier and has better anti-tissue adhesion efficiency.

Biomimetic Scaffolds for Bone Tissue Engineering.

The use of biomimetic scaffolds for bone tissue engineering has been studied for a long time. Biomimetic scaffolds can assist and accelerate bone regeneration that is similar to that of authentic tissue, which represents the environment of cells in a living organism. Currently, numerous biomaterials have been reported for use as a biomimetic scaffold. This review focuses on the design of biomimetic scaffolds, kinds of biomaterials and methods used to fabricate biomimetic scaffolds, growth factors used with biomimetic scaffold for bone regeneration, mobilization of biological agents into biomimetic scaffolds, and studies on (pre)clinical bone regeneration from biomimetic scaffolds. Then, future prospects for biomimetic scaffolds are discussed.

Cross-linked electrospun cartilage acellular matrix/poly(caprolactone-co-lactide-co-glycolide) nanofiber as an antiadhesive barrier.

In this work, we chose cartilage acellular matrix (CAM) as a promising antiadhesive material because CAM effectively inhibits the formation of blood vessels, and we used electrospinning to prepare antiadhesive barriers. Additionally, we synthesized N-hydroxysuccinimide (NHS)-poly(caprolactone-co-lactide-co-glycolide)-NHS (MP) copolymers (to tune degradation) as a cross-linking agent for CAM. This is the first report on the development of electrospun cross-linked (Cx) CAM/MP (CA/P) nanofiber (NF) (Cx-CA/P-NF) with a tunable degradation period as an antiadhesive barrier. Compared with the CA/P-NF before cross-linking, the electrospun Cx-CA/P-NF after cross-linking showed different biodegradation. Cx-CA/P-NF significantly inhibited the in vitro attachment and proliferation of human umbilical vein endothelial cells (HUVECs), as confirmed by an MTT assay and scanning electron microscopy images. Cx-CA/P-NFs implanted between a surgically damaged peritoneal wall and cecum gradually degraded in 7 days; this process was monitored by NIR imaging. The in vivo evaluation of the anti-tissue adhesive effect of Cx-CA/P-NFs revealed little adhesion, few blood vessels, and negligible inflammation at 7 days determined by hematoxylin and eosin staining. ED1 staining of Cx-CA/P-NFs showed infiltration of few macrophages because of the inflammatory response to the Cx-CA/P-NF as compared with an untreated injury model. Additionally, Cx-CA/P-NFs significantly suppressed the formation of blood vessels between the peritoneal wall and cecum, according to CD31 staining. Overall, Cx-CA/P-NFs yielded little adhesion, infiltration by macrophages, or formation of blood vessels in a postoperative antiadhesion assay. Thus, it is reasonable to conclude that the Cx-CA/P-NF designed herein successfully works as an antiadhesive barrier with a tunable degradation period. STATEMENT OF SIGNIFICANCE: The cartilage acellular matrix (CAM) can inhibit the formation of fibrous tissue bridges and blood vessels between the tissue at an injured site and the surrounding healthy tissues. However, CAM has not been rigorously investigated as an antiadhesive barrier. In this manuscript, the cross-linked CAM nanofiber (Cx-CA/P-NF) designed herein successfully works as an antiadhesive barrier. Cx-CA/P-NFs yielded little adhesion, infiltration by macrophages, or formation of blood vessels in a postoperative antiadhesion assay. Moreover, we demonstrated the suitable properties of Cx-CA/P-NF such as easy cross-linking by maintaining the antiadhesive properties, controllable biodegradation, and in vivo antiadhesive effect of Cx-CA/P-NF.

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.

Effect of Drug Carrier Melting Points on Drug Release of Dexamethasone-Loaded Microspheres.

Here, we examined the effect of melting point of drug carriers on drug release of dexamethasone (Dex)-loaded microspheres. We prepared poly(L-lactide-ran-ε-caprolactone) (PLC) copolymers with varying compositions of poly(ε-caprolactone) (PCL) and poly(L-lactide) (PLLA). As the PLLA content increased, the melting points of PLC copolymers decreased from 61 to 43 °C. PLC copolymers in vials solubilized at 40-50 °C according to the incorporation of PLLA into the PCL segment. Dexamethasone (Dex)-loaded PLC (MCxLy) microspheres were prepared by the oil-in-water (O/W) solvent evaporation/extraction method. The preparation yields were above 70%, and the mean particle size ranged from 30 to 90 µm. The MCxLy microspheres also showed controllable melting points in the range of 40-60 °C. Dex-loaded MCxLy microspheres showed similar in vitro and in vivo sustained release patterns after the initial burst of Dex. The in vitro and in vivo order of the Dex release was MC80L20 > MC90L10 > MC95L5, which agreed well with the melting point order of the drug carrier. Using in vivo fluorescence imaging of fluorescein (FI)-loaded microspheres implanted in animals, we confirmed the sustained release of FI over an extended period. In vivo inflammation associated with the PLC microsphere implants was less pronounced than that associated with Poly(lactide-co-glycolide) (PLGA). In conclusion, we successfully demonstrated that it is possible to control Dex release using Dex-loaded MCxLy microspheres with different melting points.