Applications of Bioactive Ions in Bone Regeneration.

The repair of large bone defects remains a huge challenge for bone regenerative medicine. To meet this challenge, a number of bone substitutes have been developed over recent years to overcome the drawbacks of traditional autograft and allograft therapies. Thus, the improvement of the osteoinductive ability of these substitutes has become a major focus for research in the field of bone tissue engineering. It has been reported that some metallic ions play an important role in bone metabolism in the human body, and that bone repair could be enhanced by incorporating these ions into bone substitutes. Moreover, it is well documented that ions released from these substitutes such as magnesium, zinc, and strontium can increase the osteogenic and angiogenic properties of bone repair scaffolds. However, the mechanisms of action of these ions on cellular bioactivity are currently unclear. Therefore, in the present article, we highlight the recent use of bioactive ions in bone tissue engineering and discuss the effects of these ions on osteogenesis and neovascularisation.

The dependence of in vivo stable ectopic chondrogenesis by human mesenchymal stem cells on chondrogenic differentiation in vitro.

In vivo niche plays an important role in determining the fate of implanted mesenchymal stem cells (MSCs) by directing committed differentiation. An inappropriate in vivo niche can also alter desired ultimate fate of exogenous MSCs even they are in vitro induced to express a specific phenotype before in vivo implantation. Studies have shown that in vitro chondrogenically differentiated MSCs are apt to lose their phenotype and fail to form stable cartilage in subcutaneous environment. We hypothesized that failure of maintaining the phenotype of induced MSCs in subcutaneous environment is due to the insufficient chondrogenic differentiation in vitro and fully differentiated MSCs can retain their chondrocyte-like phenotype and form stable ectopic cartilage. To test this hypothesis, extended in vitro chondrogenic induction and cartilage formation were carried out before implantation. Human bone marrow stem cells (hBMSCs) were seeded onto polylactic acid coated polyglycolic acid scaffolds. The cell-scaffold constructs were chondrogenically induced from 4 to 12 weeks for in vitro chondrogenesis, and then implanted subcutaneously into nude mice for 12 or 24 weeks. The engineered cartilages were evaluated by gross view, glycosaminoglycan content measurement, and histological staining before and after implantation. Histological examination showed typical cartilage structure formation after 8 weeks of induction in vitro. However, part of the constructs became ossified after implantation when in vitro induction lasted 8 weeks or less time. In contrast, those induced for 12 weeks in vitro could retain their cartilage structure after in vivo implantation. These results indicate that a fully differentiated stage achieved by extended chondrogenic induction in vitro is necessary for hBMSCs to form stable ectopic chondrogenesis in vivo.

Repair of canine mandibular bone defects with bone marrow stromal cells and porous beta-tricalcium phosphate.

Tissue engineering has become a new approach for repairing bone defects. Previous studies have been limited to the use of slow-degradable scaffolds with bone marrow stromal cells (BMSCs) in mandibular reconstruction. In this study, a 30 mm long mandibular segmental defect was repaired by engineered bone graft using osteogenically induced autologous BMSCs seeded on porous beta-tricalcium phosphate (beta-TCP, n=5). The repair of defects was compared with those treated with beta-TCP alone (n=6) or with autologous mandibular segment (n=4). In the BMSCs/beta-TCP group, new bone formation was observed from 4 weeks post-operation, and bony-union was achieved after 32 weeks, which was detected by radiographic and histological examination. In contrast, minimal bone formation with almost fibrous connection was observed in the group treated with beta-TCP alone. More importantly, the engineered bone with BMSCs/beta-TCP achieved a satisfactory biomechanical property in terms of bending load strength, bending displacement, bending stress and Young's modulus at 32 weeks post-operation, which was very close to those of contralateral edentulous mandible and autograft bone (p>0.05). Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and biodegradable beta-TCP can well repair the critical-sized segmental mandibular defects in canines.

[Potential of chondrogenesis of bone marrow stromal cells co-cultured with chondrocytes on biodegradable scaffold: in vivo experiment with pigs and mice].

OBJECTIVE: To explore the feasibility of in vivo chondrogenesis of bone marrow stromal cells (BMSCs) co-cultured with chondrocytes on biodegradable scaffold. METHODS: Porcine BMSCs were isolated, expanded and labeled with enhanced green fluorescent protein (EGFP), and then were mixed with articular chondrocytes isolated from porcine knee joint at the ratio of 1:1. The mixed cells were seeded onto polyglycolic acid (PGA) scaffold at the ultimate concentration of 5.0 x 10(7)/ml (co-culture group). Pure chondrocytes and BMSCs of the same ultimate concentration were seeded respectively onto the scaffold as positive control group and negative control group. After two weeks' culture in vitro, they were planted subcutaneously into nude mice respectively. These specimens were collected after in vivo implantation for 8 weeks to undergo microscopy. Laser confocal microscopy was used to observe the distribution of EGFP-labeled cells in the tissue. RT-PCR was used to examine the expression of collagen type II and aggrecan. Immunohistochemistry was used to observe the protein expression of collagen type II. RESULTS: The cell-scaffold constructs of the co-culture group and positive control group, could maintain the original size and shape no matter in vitro or in vivo. After 8 weeks' in vivo implantation, the constructs in both co-culture group and positive control group formed cartilage-like tissue with typical histological structure and extracellular matrix staining similar to those of the normal cartilage. The GAG content and compressive modulus of the co-culture group reached over 80% of those of the positive control group. Confocal microscopy revealed the presence of EGFP-labeled cells in the engineered cartilage lacuna. Histological examination showed that the constructs of the negative control group shrunk gradually after in vivo implantation with no typical cartilage-like tissue formation. CONCLUSION: In vitro co-cultured BMSC-chondrocyte-PGA constructs have the potential to form mature cartilage-like tissue in subcutaneous non-chondrogenesis environment, indicating that chondrocytes still provide enough signals for BMSC chondrogenic differentiation.

Treating metastatic triple negative breast cancer with CD44/neuropilin dual molecular targets of multifunctional nanoparticles.

Metastasis of cancer makes up the vast majority of cancer-related deaths, and it usually initiates from tumor cells invasiveness and develops through tumor neovasculature. In this work, we have fabricated a CD44/neuropilin dual receptor-targeting nanoparticulate system (tLyP-1-HT NPs) with endogenous or FDA approved components for treating metastatic triple negative breast cancer (TNBC). The enhanced specific targeting of tLyP-1-HT NPs to both metastatic tumor cells and metastasis-supporting tumor neovasculature was contributed by means of CD44/neuropilin dual receptor-mediated interaction. The NPs not only effectively suppress the invasive capability of tumor cells themselves, but also significantly restrain the metastasis incidence via extravasation as well as the eventual colonization in lungs. In all the three types of TNBC-bearing mice models, orthotopic, post-metastasis and metastasis prevention models, the docetaxel-loaded tLyP-1-HT NPs exhibited markedly enhanced anti-tumor and anti-metastasis efficacy. The inhibitory rates of tLyP-1-HT NPs against orthotopic tumor growth and lung metastasis achieved 79.6% and 100%, respectively. The metastasis inhibition rate and life extension rate of the tLyP-1-HT NPs against post-pulmonary metastasis mice reached 85.1% and up to 62.5%, respectively. All the results demonstrated the designed dual receptor-targeting multifunctional NPs hold great potential in treating metastatic TNBC and lung metastasis.

Prefabricated, ear-shaped cartilage tissue engineering by scaffold-free porcine chondrocyte membrane.

BACKGROUND: Ear defects caused by traumatic injury, tumor ablation, and congenital deficiency are still challenging problems for the plastic and reconstructive surgeon. The authors developed a scaffold-free, ear-shaped cartilage by tailoring a multilayered chondrocyte membrane on an ear-shaped titanium alloy model and investigated the possibility of long-term ear-shaped maintenance in nude mice. METHODS: High-density chondrocytes (approximately 30 × 10 cells) were seeded to produce chondrocyte membranes after cultivation under chondrogenic medium for 2 weeks. Then, three-layer chondrocyte membranes were tailored on the ear-shaped titanium mold and fixed by 6-0 nylon. The constructs were implanted onto the dorsal pockets of nude mice for 8 and 24 weeks. The chondrocyte membrane, 8- and 24-week implants were analyzed by safranin O, toluidine blue, elastica van Gieson, and collagen type II immunohistochemistry stains and quantitative measurement of glycosaminoglycan and total collagen compared with native cartilage. Mechanical strength was compared by compressive Young's modulus. RESULTS: Results showed that the chondrocyte membrane was durable and nonfragile and easily manipulated by forceps. The composite of chondrocyte membrane and titanium alloy maintained the stable ear-like shape after 8 and 24 weeks of subcutaneous implantation. Histologic examination verified that the newly formed tissue at the implant construct was elastic cartilage at both 8 and 24 weeks by safranin O, toluidine blue, elastica van Gieson, and collagen type II immunohistochemistry stains. The Young's modulus was only half of and similar to normal cartilage in 8- and 24-week implants, respectively. CONCLUSION: This study demonstrated that an ear-shaped elastic cartilage could be regenerated by a scaffold-free chondrocyte membrane shaped by a prefabricated, three-dimensional, ear-shaped titanium mold.

Repair of Achilles tendon defect with autologous ASCs engineered tendon in a rabbit model.

Adipose derived stem cells (ASCs) are an important cell source for tissue regeneration and have been demonstrated the potential of tenogenic differentiation in vitro. This study explored the feasibility of using ASCs for engineered tendon repair in vivo in a rabbit Achilles tendon model. Total 30 rabbits were involved in this study. A composite tendon scaffold composed of an inner part of polyglycolic acid (PGA) unwoven fibers and an outer part of a net knitted with PGA/PLA (polylactic acid) fibers was used to provide mechanical strength. Autologous ASCs were harvested from nuchal subcutaneous adipose tissues and in vitro expanded. The expanded ASCs were harvested and resuspended in culture medium and evenly seeded onto the scaffold in the experimental group, whereas cell-free scaffolds served as the control group. The constructs of both groups were cultured inside a bioreactor under dynamic stretch for 5 weeks. In each of 30 rabbits, a 2 cm defect was created on right side of Achilles tendon followed by the transplantation of a 3 cm cell-seeded scaffold in the experimental group of 15 rabbits, or by the transplantation of a 3 cm cell-free scaffold in the control group of 15 rabbits. Animals were sacrificed at 12, 21 and 45 weeks post-surgery for gross view, histology, and mechanical analysis. The results showed that short term in vitro culture enabled ASCs to produce matrix on the PGA fibers and the constructs showed tensile strength around 50 MPa in both groups (p > 0.05). With the increase of implantation time, cell-seeded constructs gradually form neo-tendon and became more mature at 45 weeks with histological structure similar to that of native tendon and with the presence of bipolar pattern and D-periodic structure of formed collagen fibrils. Additionally, both collagen fibril diameters and tensile strength increased continuously with significant difference among different time points (p < 0.05). In contrast, cell-free constructs failed to form good quality tendon tissue with fibril structure observable only at 45 weeks. There were significant differences in both collagen fibril diameter and tensile strength between two groups at all examined time points (p < 0.05). The results of this study support that ASCs are likely to be a potential cell source for in vivo tendon engineering and regeneration.

Engineering ear-shaped cartilage using electrospun fibrous membranes of gelatin/polycaprolactone.

Tissue engineering approach continuously requires for emerging strategies to improve the efficacy in repairing and regeneration of tissue defects. Previously, we developed a sandwich model strategy for cartilage engineering, using the combination of acellular cartilage sheets (ACSs) and chondrocytes. However, the process for the preparation of ACSs is complicated, and it is also difficult to obtain large ACSs. The aim of this study was to engineer cartilage with precise three-dimensional (3-D) structures by applying electrospun fibrous membranes of gelatin/polycaprolactone (GT/PCL). We first prepared the electrospun GT/PCL membranes into rounded shape, and then seeded chondrocytes in the sandwich model. After in vitro and in vivo cultivation, the newly formed cartilage-like tissues were harvested. Macroscopic observations and histological analysis confirmed that the engineering of cartilage using the electrospun GT/PCL membranes was feasible. An ear-shaped cartilage was then constructed in the sandwich model, with the help of an ear-shaped titanium alloy mold. After 2 weeks of culture in vitro and 6 weeks of subcutaneous incubation in vivo, the ear-shaped cartilage largely maintained their original shape, with a shape similarity up to 91.41% of the titanium mold. In addition, the engineered cartilage showed good elasticity and impressive mechanical strength. These results demonstrated that the engineering of 3-D cartilage in a sandwich model using electrospun fibrous membranes was a facile and effective approach, which has the potential to be applied for the engineering of other tissues with complicated 3-D structures.

Engineering of epidermis skin grafts using electrospun nanofibrous gelatin/ polycaprolactone membranes.

Skin engineering provides a new strategy for treating a wide variety of skin defects. In particular, electrospun nanofibrous membranes have been used as carriers for epidermis engineering. The aim of this study was to investigate the feasibility of a modified gelatin and polycaprolactone (GT/PCL) electrospun membrane for epidermis engineering. The biocompatibility of the membranes was evaluated by seeding HaCaT cells (human keratinocyte cell line) on the membrane and the mechanical properties of the membranes were determined with and without cells after culture. A cell proliferation assay showing that HaCaT cells attached and proliferated well on the membranes demonstrated that the membranes possess good biocompatibility. Mechanical tests showed that the membranes are strong enough to be handled during transplantation. Further in vivo transplantation studies revealed that epidermises engineered with GT/PCL membranes were able to repair skin defects in the nude mouse. These results demonstrate that GT/PCL electrospun membranes could be suitable scaffolds for skin engineering.

[Repair of calvarial defects with human umbilical cord blood derived mesenchymal stem cells and demineralized bone matrix in athymic rats].

OBJECTIVE: To investigate the feasibility of using human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) and demineralized bone matrix (DBM) scaffolds to repair critical-sized calvarial defects in athymic rats. METHODS: Human UCB-MSCs were isolated, expanded and osteogenically induced in vitro. Osteogenic differentiation of UCB-MSCs was evaluated by Alizarin Red staining and measurement of calcium content respectively, and then the cells were seeded onto DBM scaffolds. Bilateral full-thickness defects (5 mm in diameter) of parietal bone were created in an athymic rat model. The defects were either repaired with UCB-MSC/DBM constructs (experimental group) or with DBM scaffolds alone (control group). Animals were harvested at 6 and 12 weeks post-implantation respectively, and defect repair was evaluated with gross observation, micro-CT measurement and histological analysis. RESULTS: Micro-CT showed that new bone was formed in the experimental group at 6 weeks post-implantation, while no sign of new bone formation was observed in the control group. At 12 weeks post-transplantation, scaffolds had been degraded almost completely in both sides. It was shown that an average of (78.19 +/- 6.45)% of each defect volume had been repaired in experimental side; while in the control side, only limited bone formed at the periphery of the defect. Histological examination revealed that the defect was repaired by trabecular bone tissue in experimental side at 12 weeks, while only fibrous connection was observed in the control group. CONCLUSIONS: Tissue-engineered bone composed of osteogenically-induced human UCB-MSCs on DBM scaffolds could successfully repair the critical-sized calvarial defects in athymic rat models.

Repair of canine mandibular bone defects with bone marrow stromal cells and coral.

Tissue engineering has become a new approach for repairing bone defects. Previous studies indicated that coral scaffolds had been utilized with bone marrow stromal cells (BMSCs) in a variety of approaches for bony reconstruction. In these applications, the degradation rate of the material did not match the rate at which bone was regenerated. In this study, a previously established 30 mm long mandibular segmental defect was repaired with engineered bone using green fluorescent protein-labeled osteogenic BMSCs seeded on porous coral (n = 12). Defects treated with coral alone (n = 12) were used as an experimental control. In the BMSCs/coral group, new bone formation was observed from 4 weeks postoperation, and bony-union was achieved after 32 postoperative weeks. The residual coral volume of the BMSCs/coral grafts at 12 weeks (20-30%) was significantly higher than that at 32 weeks (10-15%, p < 0.05), which was detected by microcomputed tomography and histological examination. The engineered bone with BMSCs/coral achieved satisfactory biomechanical properties at 32 weeks postoperation, which was very close to that of the contralateral edentulous mandible. More importantly, immunostaining demonstrated that the implanted BMSCs differentiated into osteoblast-like cells. In contrast, minimal bone formation with almost solely fibrous connection was observed in the group treated with coral alone. Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and biodegradable coral can successfully repair the critical-sized segmental mandibular defects in canines and the seeding cells could be used for bony restoration.

In vivo engineering of a functional tendon sheath in a hen model.

Repair of injured tendon sheath remains a major challenge and this study explored the possibility of in vivo reconstruction of a tendon sheath with tendon sheath derived cells and polyglycolic acid (PGA) fibers in a Leghorn hen model. Total 55 Leghorn hens with a 1cm tendon sheath defect created in the left middle toe of each animal were randomly assigned into: (1) experimental group (n=19) that received a cell-PGA construct; (2) scaffold control group (n=18) that received a cell-free PGA scaffold; (3) blank control group (n=18) with the defect untreated. Tendon sheath cells were isolated, in vitro expanded, and seeded onto PGA scaffolds. After in vitro culture for 7 days, the constructs were in vivo implanted to repair the sheath defects. Alcian blue staining confirmed the ability of cultured cells to produce specific matrices containing acidic carboxyl mucopolysaccharide (mainly hyaluronic acid). In addition, the engineered sheath formed a relatively mature structure at 12 weeks post-surgery, which was similar to that of native counterpart, including a smooth inner surface, a well-developed sheath histological structure with a clear space between the tendon and the engineered sheath. More importantly, Work of Flexion assay revealed that the tendons needed less power consumption to glide inside the engineered sheath when compared to the tendons which were surrounded by scar-repaired tissues, indicating that the engineered sheaths had gained the function to a certain extent of preventing tendon adhesion. Taken together, these results suggest that tendon sheaths that are functionally and structurally similar to native sheaths are possible to be engineered in vivo using tendon sheath cells and PGA scaffolds.

In vitro precultivation alleviates post-implantation inflammation and enhances development of tissue-engineered tubular cartilage.

Tissue-engineered tubular cartilage is a promising graft for tracheal reconstruction. But polylactic acid/polyglycolic acid (PLA/PGA) fibers, the frequently used scaffolds for cartilage engineering, often elicit an obvious inflammation response following implantation into immunocompetent animals. We propose that the inflammation could be alleviated by in vitro precultivation. In this study, after in vitro culture for either 2 days (direct implantation group (DI)) or for 2 weeks (precultivation implantation group (PI)), autologous tubular chondrocyte-PLA/PGA constructs were subcutaneously implanted into rabbits. In the PI group, after 2 weeks of precultivation, most of the fibers were found to be completely embedded in an extracellular matrix (ECM) produced by the chondrocytes. Importantly, no obvious inflammatory reaction was observed after in vivo implantation and homogeneous cartilage-like tissue was formed with biomechanical properties close to native tracheal cartilage at 4 weeks post-implantation. In the DI group, however, an obvious inflammatory reaction was observed within and around the cell-scaffold constructs at 1 week implantation and only sporadic cartilage islands separated by fibrous tissue were observed at 4 weeks. These results demonstrated that the post-implantation inflammatory reaction could be alleviated by in vitro precultivation, which contributes to the formation of satisfactory tubular cartilage for tracheal reconstruction.

Tissue engineering of corneal stromal layer with dermal fibroblasts: phenotypic and functional switch of differentiated cells in cornea.

Previously, we successfully engineered a corneal stromal layer using corneal stromal cells. However, the limited source and proliferation potential of corneal stromal cells has driven us to search for alternative cell sources for corneal stroma engineering. Based on the idea that the tissue-specific environment may alter cell fate, we proposed that dermal fibroblasts could switch their phenotype to that of corneal stromal cells in the corneal environment. Thus, dermal fibroblasts were harvested from newborn rabbits, seeded on biodegradable polyglycolic acid (PGA) scaffolds, cultured in vitro for 1 week, and then implanted into adult rabbit corneas. After 8 weeks of implantation, nearly transparent corneal stroma was formed, with a histological structure similar to that of its native counterpart. The existence of cells that had been retrovirally labeled with green fluorescence protein (GFP) demonstrated the survival of implanted cells. In addition, all GFP-positive cells that survived expressed keratocan, a specific marker for corneal stromal cells, and formed fine collagen fibrils with a highly organized pattern similar to that of native stroma. However, neither dermal fibroblast-PGA construct pre-incubated in vitro for 3 weeks nor chondrocyte-PGA construct could form transparent stroma. The results demonstrated that neonatal dermal fibroblasts could switch their phenotype in the new tissue environment under restricted conditions. The functional restoration of corneal transparency using dermal fibroblasts suggests that they could be an alternative cell source for corneal stroma engineering.

Tissue engineering of blood vessel.

Vascular grafts are in large demand for coronary and peripheral bypass surgeries. Although synthetic grafts have been developed, replacement of vessels with purely synthetic polymeric conduits often leads to the failure of such graft, especially in the grafts less than 6 mm in diameter or in the areas of low blood flow, mainly due to the early formation of thrombosis. Moreover, the commonly used materials lack growth potential, and long-term results have revealed several material-related failures, such as stenosis, thromboembolization, calcium deposition and infection. Tissue engineering has become a promising approach for generating a bio-compatible vessel graft with growth potential. Since the first success of constructing blood vessels with collagen and cultured vascular cells by Weinberg and Bell, there has been considerable progress in the area of vessel engineering. To date, tissue- engineered blood vessels (TEBVs) could be successfully constructed in vitro, and be used to repair the vascular defects in animal models. This review describes the major progress in the field, including the seeding cell sources, the biodegradable scaffolds, the construction technologies, as well as the encouraging achievements in clinical applications. The remaining challenges are also discussed.