Tianqiu Mao

Engineering scaffold‐free bone tissue using bone marrow stromal cell sheets

The use of exogenous scaffolds to engineer bone tissue faces several drawbacks including insufficient biological activity, potential immunogenicity, elevated inflammatory reaction, fluctuating degradation rate, and low cell‐attachment efficiency. To circumvent these limitations, we sought to engineer large scaffold‐free bone tissue using cell sheets. We harvested intact cell sheets from bone marrow stromal cells using a continuous culture method and a scraping technique. The cell sheets were then rolled and fabricated into large constructs. Finally, the constructs were implanted into the subcutaneous pockets of nude mice. The cells within the sheet maintained in vitro osteogenic potential after osteoblast differentiation. Computed tomography scans and histological examination confirmed new bone formation in vivo. Additionally, the engineered bone exhibited enhanced compressive strength. Our results indicate that the BMSC sheets can facilitate the formation of functional three‐dimensional bone tissue without the use of exogenous scaffolds. Hence, the study provides an intriguing alternative strategy for bone repair.

Platelet-rich plasma – A promising cell carrier for micro-invasive articular cartilage repair

Due to the limited regenerative capacity of cartilage tissues, articular cartilage defect caused by various lesions remains a problem to be resolved. Tissue engineering provided a valuable alternative to current therapeutic approaches, which is expected to greatly reduce the need of joint replacement. Scaffold, acting as cell carrier, plays an important role in maintaining cells in defect sites, thus facilitates the chondrogenesis. However, an open operation is often needed to implant the cell/scaffold composite, to find a less invasive way to delivering the complex into the defect site would be desirable. Different from synthetic and other nature derived scaffold, platelet-rich plasma (PRP) is a fraction of plasma which contains multiple growth factors and could be clotted when mixed with thrombin. Therefore, we hypothesized that PRP could be used as an autologous cell carrier to inject and fix chondrocytes into the defect site of articular cartilage. With the assistance of arthroscope, the defect could be precisely located, and injectable PRP-Cell composite would make the operation micro-invasive and simple.

Vitalisation of tubular coral scaffolds with cell sheets for regeneration of long bones: a preliminary study in nude mice

In this study, cell sheets comprising multilayered living bone marrow stromal cells and extracellular matrix were assembled with tubular coral scaffolds for long bone regeneration. Cell sheet with visible mineralized nodules was harvested and wrapped against tubular coral scaffolds with 5mm diameter and 1.5mm wall thickness. New bone formation was investigated by CT scan and histological observation 8 and 12 weeks after implantation of cell sheet/scaffold. The results showed that cortical bone formed within the constructs for both groups. New bone composed 25.75% of the graft in 8 weeks group, compared to that of 40.01% in 12 weeks group. Histological examination showed that new bone formation was in the manner of endochondral osteogenesis, with woven bone matrix subsequently maturing into fully mineralized compact bone. These findings demonstrated that osteogenic cell sheet could vitalize tubulate coral scaffolds to regenerate bone graft with similar shape and structure to native bone.

Enhancing bone formation by transplantation of a scaffold‐free tissue‐engineered periosteum in a rabbit model

OBJECTIVES The periosteum plays an important role in bone regeneration. However, the harvesting of autogenous periosteum is associated with disadvantages such as donor site morbidity and limited donor sources. This study uses an osteogenic predifferentiated cell sheet to fabricate a scaffold-free tissue-engineered periosteum (TEP). MATERIAL AND METHODS We generated an osteogenic predifferentiated cell sheet from rabbit bone marrow stromal cells (BMSCs) using a continuous culture system and harvested it using a scraping technique. Then, the in vitro characterization of the sheet was investigated using microscopy investigation, quantitative analysis of alkaline phosphatase (ALP) activity, and RT-PCR. Next, we demonstrated the in vivo osteogenic potential of the engineered sheet in ectopic sites together with a porous β-tricalcium phosphate ceramic. Finally, we evaluated its efficiency in treating delayed fracture healing after wrapping the cell sheet around the mandible in a rabbit model. RESULTS The engineered periosteum showed sporadic mineralized nodules, elevated ALP activity, and up-regulated gene expression of osteogenic markers. After implantation in the subcutaneous pockets of the donor rabbits, the in vivo bone-forming capability of the engineered periosteum was confirmed by histological examinations. Additionally, when wrapping the engineered periosteum around a mandibular fracture gap, we observed improved bone healing and reduced amounts of fibrous tissue at the fracture site. CONCLUSION The osteogenic predifferentiated BMSC sheet can act as a scaffold-free TEP to facilitate bone regeneration. Hence, our study provides a promising strategy for enhancing bone regeneration in clinical settings.

Engineering injectable bone using bone marrow stromal cell aggregates.

With the increasing popularity of minimally invasive surgery, to develop an injectable bone would be highly preferable for the repair of bone nonunions and defects. However, the use of dissociated cells and exogenous carriers to construct injectable bone faces several drawbacks. To circumvent these limitations, we first harvested a cell sheet from rabbit bone marrow stromal cells using a continuous culture method and a scraping technique. The obtained sheet was then cut into fragments of multicellular aggregates, each of which was composed of a certain number of cells, extracellular matrix, and intercellular connections. The aggregates showed apparent mineralization properties, high alkaline phosphatase activity, increased osteocalcin content, and upregulated bone markers, implying their in vitro osteogenic potential. Then, serum-free medium (the control group), dissociated cell suspension (the cell group), and suspension of multicellular aggregates (the aggregate group) were injected subcutaneously on the back of the nude mice to evaluate ectopic bone formation. The results revealed that the aggregate group showed significantly larger and denser bone at the injection sites than the cell group, whereas bone formation did not occur in the control group. Additionally, when injecting them locally into the mandibular fracture gap of delayed healing in a rabbit model, we observed the most improved bone healing in the aggregate group. More cells survive and retain at the injection sites in the aggregate group than that in the cell group postoperatively. Our study indicates that the multicellular aggregates might be considered a promising strategy to generate injectable bone tissue and improve the efficacy of cell therapy.

The role of the lateral pterygoid muscle in the sagittal fracture of mandibular condyle (SFMC) healing process

The aim of this study was to examine the role of the lateral peterygoid muscle in the reconstruction of the shape of the condyle during healing of a sagittal fracture of the mandibular condyle. Twenty adult sheep were divided into 2 groups: all had a unilateral operation on the right side when the anterior and posterior attachments of the discs were cut, and an oblique vertical osteotomy was made from the lateral pole of the condyle to the medial side of the condylar neck. Ten sheep had the lateral pterygoid muscle cut, and the other 10 sheep did not. Sheep were killed at 4 weeks (n=2 from each group), 12 weeks (n=4), and 24 weeks (n=4) postoperatively. Computed tomograms (CT) were taken before and after operations. We dissected the joints, and recorded with the naked eye the shape, degree of erosion, and amount of calcification of the temporomandibular joint (TMJ). In the group in which the lateral peterygoid muscle had not been cut the joints showed overgrowth of new bone and more advanced ankylosis. Our results show that the lateral pterygoid muscle plays an important part in reconstructing the shape of the condyle during the healing of a sagittal fracture of the mandibular condyle, and combined with the dislocated and damaged disc is an important factor in the aetiology of traumatic ankylosis of the TMJ.

Reconstruction of rabbit critical-size calvarial defects using autologous bone marrow stromal cell sheets.

The reconstruction of bone defects remains a significant clinical problem. In this study, we constructed cell sheet from bone marrow stromal cells on normal culture plates by a simple method. The cell sheets showed evident mineralized nodules, high alkaline phosphatase activities, indicating their in vitro osteogenic potential. Then its osteogenic capability to heal critical-size rabbit calvarial defect was investigated. Forty adult New Zealand White rabbits were randomly divided into 4 groups of 10 animals each: (1) empty, (2) demineralized bone matrix (DBM) alone, (3) DBM/cell suspension, and (4) DBM/cell sheet. Specimens were harvested 6 and 12 weeks after implantation, respectively. Radiographic, histologic, and histomorphometric analyses were performed to evaluate the new bone formation inside the defect. The results revealed that the defect treated with DBM/cell sheet showed significantly more bone formation than other 3 groups (P < 0.05). Our study indicates that the cell sheet enhances bone regeneration in healing critical-size rabbit calvarial defect, and cell sheet-based engineered bone might be considered as potential substitutes for bone reconstruction.

Locally injection of cell sheet fragments enhances new bone formation in mandibular distraction osteogenesis: A rabbit model

Effective methods to shorten the treatment period of distraction osteogenesis (DO) are needed. To investigate whether injections of osteogenic bone marrow stromal cell (BMSC) sheet fragments could be used to facilitate new bone formation during DO, 30 rabbits underwent bilateral mandibular osteotomy and their mandibles were lengthened at a rate of 0.75 mm/12 h for 6 days after a 5‐day latency period. There were three treatment groups (n = 10 for each group): Serum‐free medium, dissociated BMSCs, and BMSC sheet fragments. A local injection was conducted with a needle directly into the distracted areas immediately after distraction. Rabbits were sacrificed for examination at 3 and 6 weeks after injection. Gross examination, radiographic evaluation, and micro‐CT scanning indicated a significant increase in bony union in the BMSC sheet fragment group, compared with the medium group and the dissociated cell group. The histomorphometric analysis showed more intensive bone formation in the sheet fragment group than the other two groups at each time point. Additionally, the peak load was significantly higher in the fragment group than those in the others. The results show that injection of BMSC sheet fragments promotes bone formation in DO and indicate a promising approach to shorten the treatment period of osteodistraction.

Prefabrication of axial vascularized tissue engineering coral bone by an arteriovenous loop: A better model

The most important problem for the survival of thick 3-dimensional tissues is the lack of vascularization in the context of bone tissue engineering. In this study, a modified arteriovenous loop (AVL) was developed to prefabricate an axial vascularized tissue engineering coral bone in rabbit, with comparison of the arteriovenous bundle (AVB) model. An arteriovenous fistula between rabbit femoral artery and vein was anastomosed to form an AVL. It was placed in a circular side groove of the coral block. The complex was wrapped with an expanded-polytetrafluoroethylene membrane and implanted beneath inguinal skin. After 2, 4, 6 and 8 weeks, the degree of vascularization was evaluated by India ink perfusion, histological examination, vascular casts, and scanning electron microscopy images of vascular endangium. Newly formed fibrous tissues and vasculature extended over the surfaces and invaded the interspaces of entire coral block. The new blood vessels robustly sprouted from the AVL. Those invaginated cavities in the vascular endangium from scanning electron microscopy indicated vessels sprouted pores. Above indexes in AVL model are all superior to that in AVB model, indicating that the modified AVL model could more effectively develop vascularization in larger tissue engineering bone.

Influence of platelet-rich plasma on ectopic bone formation of bone marrow stromal cells in porous coral

This study evaluated the effect of platelet-rich plasma (PRP) on the bone formation of marrow stromal cells (MSCs) in porous coral. MSCs in 50 μl of PRP were seeded into natural coral disks (diameter 8.0 mm; thickness 2.0 mm). The composites were clotted and cultured in vitro or implanted subcutaneously into nude mice. Coral scaffolds loading MSCs or PRP alone acted as control. Alkaline phosphatase (ALP) activity of the specimens cultured in vitro for 7 and 14 days was measured, and the level of ectopic bone formation was investigated 4 and 8 weeks after operation. The samples from the coral/PRP/MSC group exhibited significantly higher ALP activity, compared with that from the coral/MSC group or the coral/PRP group (p<0.05). New bone and/or cartilage formation could be observed in specimens from both coral/PRP/MSC and coral/MSC groups in ectopic sites, and osteogenesis followed the pattern of endochondral bone formation. Histomorphometric analyses showed enhanced cartilage and/or bone formation in the coral/PRP/MSC group, 4 and 8 weeks after implantation. No bone or cartilage formation could be observed in the coral/PRP group. The authors concluded that PRP could improve the ALP activity of MSCs on coral and increase ectopic bone formation.