Caiwen Xiao

Bone marrow stromal cells with a combined expression of BMP-2 and VEGF-165 enhanced bone regeneration*

Bone graft substitutes with osteogenic factors alone often exhibit poor bone regeneration due to inadequate vascularization. Combined delivery of osteogenic and angiogenic factors from biodegradable scaffolds may enhance bone regeneration. We evaluated the effects of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF), combined with natural coral scaffolds, on the repair of critical-sized bone defects in rabbit orbits. In vitro expanded rabbit bone marrow stromal cells (BMSCs) were transfected with human BMP2 and VEGF165 genes. Target protein expression and osteogenic differentiation were confirmed after gene transduction. Rabbit orbital defects were treated with a coral scaffold loaded with BMP2-transduced and VEGF-transduced BMSCs, BMP2-expressing BMSCs, VEGF-expressing BMSCs, or BMSCs without gene transduction. Volume and density of regenerated bone were determined by micro-computed tomography at 4, 8, and 16 weeks after implantation. Neovascularity, new bone deposition rate, and new bone formation were measured by immunostaining, tetracycline and calcein labelling, and histomorphometric analysis at different time points. The results showed that VEGF increased blood vessel formation relative to groups without VEGF. Combined delivery of BMP2 and VEGF increased new bone deposition and formation, compared with any single factor. These findings indicate that mimicking the natural bone development process by combined BMP2 and VEGF delivery improves healing of critical-sized orbital defects in rabbits.

In Vivo Efficacy of Bone Marrow Stromal Cells Coated with Beta-Tricalcium Phosphate for the Reconstruction of Orbital Defects in Canines

PURPOSE To repair the segmental orbital rim defects of dogs with three-dimensional (3D) tissue-engineered constructs derived from culturing autogenous bone marrow stromal cells (BMSCs) on β-tricalcium phosphate (β-TCP) scaffolds. METHODS A 25-mm segmental defect on the canine inferior orbital rim was created. BMSCs were isolated and osteogenically induced in vitro, then were seeded onto 3D β-TCP scaffolds and implanted to repair the orbital defects after 5 days of cultivation. The group of noninduced BMSC/β-TCP, β-TCP alone, and the normal inferior orbital rim were set as controls. The orbits of all groups had spiral computed tomography (CT) scans 1, 4, 8, and 12 weeks after surgery. Gross examination, bone density, microCT, and histologic measurements were performed 12 weeks after surgery. The results were analyzed to evaluate the extent of bone repair. RESULTS Twelve weeks after surgery, CT examination revealed good inferior orbital rim recovery in the induced BMSC/β-TCP group, and the bone density was 0.30 ± 0.03 g/cm(2) with no dominant variance, compared with the normal control (P > 0.05). MicroCT and histologic examination confirmed that the implantations led to good repair of the defects. Pore-like spongy bone surrounded the implants through the section plane, with some residue remaining in the center. In contrast, the noninduced BMSC/β-TCP implants were not fully repaired, and nonunion was evident. The bony density for this group was 0.23 ± 0.07 g/cm(2), which was significantly lower than that of the control group (P < 0.05). The β-TCP group was largely held by fibrous tissues. CONCLUSIONS Engineered bone from induced BMSCs and 3D biodegradable β-TCP can efficiently repair critical-sized segmental orbital defects in dogs.

In vitro osteogenic induction of bone marrow stromal cells with encapsulated gene-modified bone marrow stromal cells and in vivo implantation for orbital bone repair.

Osteogenic induction with either growth factors or genetic modification has limitations due to the short half-life and cost of the former, or safety concerns regarding the latter. The objective of this study was to employ a microcapsulation technique to separate genetically modified and nonmodified bone marrow stromal cells (BMSCs) to establish a cost-effective and biosafe osteogenic induction methodology with functional evaluation in vitro and in vivo in a canine model. Autologous BMSCs were isolated and transduced with adenoviral vectors containing either BMP-2 or vascular endothelial growth factor (VEGF) or were dual transduced followed by encapsulation in alginate microcapsules using an electrostatic bead generator. After cocultured with encapsulated cells, normal autologous BMSCs were analyzed for osteogenic differentiation and seeded onto tricalcium phosphate (TCP) scaffolds for in vivo implantation to repair orbital wall bone defects (12 mm in diameter) in a canine model. In vitro assays showed that the expression of the transduced genes was significantly upregulated, with significantly more transduced proteins released from the transduced cells compared with control cells. Importantly, examination of the BMSCs induced by soluble factors released from the encapsulated cells revealed a significant upregulation of expression of osteogenic markers Runx2, BSP, OPN, and OCN in dual-transduction or induction groups. In addition, dual transduction and induction resulted in the highest increase of alkaline phosphatase activity and mineralization compared with other experimental groups. In vivo assays using CT, micro-CT, and histology further supported the qPCR and western blot findings. In conclusion, encapsulation of genetically modified BMSCs was able to release a sufficient amount of BMP-2 and VEGF, which effectively induced osteogenic differentiation of normal-cultured BMSCs and demonstrated bone repair of the orbital wall defect after implantation with β-TCP in vivo.

Palpebral lipogranuloma caused by transcanalicular ointment injection after laser canaliculoplasty.

Purpose: Laser canaliculoplasty is widely used to treat lacrimal duct obstruction. We describe 12 patients in whom eyelid lipogranuloma developed subsequent to laser canaliculoplasty, followed by tobramycin and dexamethasone, ofloxacin, or erythromycin ointment injected in the lacrimal duct. Methods: Clinical history, ocular workup, and CT were performed. Histopathologic examination was performed after eyelid tissue excision. Results: Over a period of 3 years, 12 patients were diagnosed with eyelid lipogranuloma caused by diffusion and deposition of ointment. Seven of 12 patients were injected with tobramycin and dexamethasone ointment, 3 patients were injected with ofloxacin ointment, and 2 patients were injected with erythromycin ointment. Histopathologic features of the excised tissue included multifocal spaces surrounded by multinucleated giant cells, considered to be lipogranulomatous inflammation. Conclusions: The incidence of lipogranuloma caused by transcanalicular ointment injection after laser canaliculoplasty is low. Surgical excision of the lipogranuloma should be performed only when it becomes relatively localized and has passed the acute phase of inflammation to avoid the recurrence of lipogranuloma and diffusion of the inflammation. A cosmetic outcome should be considered, and simultaneous blepharoplasty with transplantation of herniated fat pads may be performed in either or both eyelids to achieve a satisfactory appearance. Injection of ointment in the lacrimal duct should be avoided, as it might create unnecessary complications when a new channel is created or when canalicular walls are penetrated. Local massage after ointment injection is not recommended.

Late correction of orbital-zygomatic-maxillary fractures combined with orbital wall fractures.

Purpose With the increasing number of patients with facial trauma, orbital-zygomatic-maxillary (OZM) fracture combined with orbital wall fracture has become common. Such fractures always induce symptoms of enophthalmos, hypoglobus, and diplopia. This study was aimed to investigate the surgical technique and operative effect of late reconstruction of OZM fractures combined with orbital wall fractures. Methods A retrospective analysis of the 46 patients (46 orbits) who were treated from January 2005 to December 2008 was performed. All cases had reconstruction of OZM fractures combined with orbital wall fractures at an average of 5.3 months after injury. Various incisions were selected. According to the computer-aided design/computer-aided manufacturing design, the osteotomy, reposition, and 4-point fixation of the fractured bones were individualized performed. Titanium mesh was preshaped according to the mirroring technology and inserted into the orbit with or without high-density polyethylene (HDPE) implant material to repair the orbital wall defect and the enlarged orbital volume. All patients were followed up 1 year after surgery. Results Forty-two of the 46 patients who had enophthalmos were completely corrected. Forty patients who had hypoglobus were corrected postoperatively. Of the 43 cases with diplopia, 20 cases were resolved. Conclusions Late reconstruction by osteotomy and 4-point fixation could recover midface cosmesis, correct the enophthalmos, and improve the diplopia of OZM fractures combined with orbital wall fractures. The computer-aided design/computer-aided manufacturing system and mirroring technology can help improve the outcome of surgery when repairing such fractures.

Repair of orbital bone defects in canines using grafts of enriched autologous bone marrow stromal cells

BackgroudBone tissue engineering is a new approach for the repair of orbital defects. The aim of the present study was to explore the feasibility of tissue-engineered bone constructed using bone marrow stromal cells (BMSCs) that were rapidly isolated and concentrated from bone marrow (BM) by the red cell lysis method, then combined with β-tricalcium phosphate (β-TCP) to create grafts used to restore orbital bone defects in canines.MethodsIn the experimental group, grafts were constructed using BMSCs obtained by red cell lysis from 20 ml bone marrow, combined with β-TCP and BM via the custom-made stem cell-scaffold device, then used to repair 10 mm diameter medial orbital wall bony defects in canines. Results were compared with those in groups grafted with BM/β-TCP or β-TCP alone, or with defects left untreated as controls. The enrichment of BMSCs and nucleated cells (NCs) in the graft was calculated from the number in untreated bone marrow and in suspensions after red cell lysis. Spiral computed tomography (CT) scans were performed 1, 4, 12 and 24 weeks after implantation in all groups. Gross examination, micro-CT and histological measurements were performed 24 weeks after surgery. The results were analyzed to evaluate the efficacy of bone repair.ResultsThe number of NCs and of colony-forming units within the scaffolds were increased 54.8 times and 53.4 times, respectively, compared with untreated bone marrow. In the BMSC-BM/β-TCP group, CT examination revealed that the scaffolds were gradually absorbed and the bony defects were restored. Micro-CT and histological examination confirmed that the implantations led to good repair of the defects, with 6 out 8 orbital defects completely restored in the experimental group, while by contrast, the grafts in the control groups did not fully repair the bony defects, a difference which was statistically significant (p < 0.05).ConclusionsTissue-engineered bone, constructed using BMSCs isolated by red cell lysis of BM, can restore critical-sized orbital wall defects in canines.

Orbital wall repair in canines with beta-tricalcium phosphate and induced bone marrow stromal cells.

Bone tissue engineering is a new approach for the repair of orbital defects. The aim of the present study was to evaluate prefabricated beta-tricalcium phosphate (β-TCP) combined with autologous bone marrow stromal cells (BMSCs) to repair orbital wall defect in canine models. Defects measuring 10 mm in diameter were created in the orbital medial walls of 12 dogs. The orbits were randomly divided into five groups: group 1, repaired with osteogenesis-induced BMSCs/TCP constructs; group 2, repaired with noninduced BMSCs/TCP constructs; group 3, repaired with β-TCP scaffolds only; group 4, normal group; group 5, negative control (bone defect without treatment). Computed tomography (CT) scanning, gross observation, bone density measurements, micro-CT, and histological observations were performed. In group 1, new bone was observed with only a small amount of residual material, and bony union was achieved 3 months after surgery. In contrast, the constructs showed slow degradation with minimal bone formation in groups 2 and 3. Furthermore, the appearance and bone density of the constructs in group 1 were similar to that of normal bone: the constructs were covered with complete mucosa, and new alveolate plate grew into the ethmoidal sinuses. A large bone defect remained in group 5. This study demonstrated that biologic scaffolds composed of β-TCP and osteogenesis-induced BMSCs have been successfully used to restore bone functionality in animal models, which may provide a potential clinical approach for orbital wall repair and bone regeneration in humans.

Spontaneous Recovery of a Fronto-Orbital Blow-in Fracture in a Child

Forehead trauma is one of the most common childhood injuries. Cranial, frontal, and orbital fractures are more ommon than midfacial and mandible fractures in chilren aged under 5 years, because the forehead prorudes, and when blunt frontal trauma occurs, the foreead absorbs the full force of the initial impact, possibly eading to linear or depressed fronto-orbital fractures. Rare cases of spontaneous resolution of depressed skull fractures, referred to as “ping-pong” or “green-stick” fractures, with inward buckling of the bone surface without loss of bone continuity have been reported in neonates, infants, and younger children. There are, however, no previous reports of spontaneous reduction of depressed facial or cranial fractures with obvious bone disruption and displacement. We present an intriguing case of a fronto-orbital blow-in fracture in a child, in which inward displacement of fragments reduced spontaneously without treatment.

Enhanced bioactivity and osteoinductivity of carboxymethyl chitosan/nanohydroxyapatite/graphene oxide nanocomposites

Tissue engineering approaches combine a bioscaffold with stem cells to provide biological substitutes that can repair bone defects and eventually improve tissue functions. The prospective bioscaffold should have good osteoinductivity. Surface chemical and roughness modifications are regarded as valuable strategies for developing bioscaffolds because of their positive effects on enhancing osteogenic differentiation. However, the synergistic combination of the two strategies is currently poorly studied. In this work, a nanoengineered scaffold with surface chemistry (oxygen-containing groups) and roughness (Rq = 74.1 nm) modifications was fabricated by doping nanohydroxyapatite (nHA), chemically crosslinked graphene oxide (GO) and carboxymethyl chitosan (CMC). The biocompatibility and osteoinductivity of the nanoengineered CMC/nHA/GO scaffold was evaluated in vitro and in vivo, and the osteogenic differentiation mechanism of the nanoengineered scaffold was preliminarily investigated. Our data demonstrated that the enhanced osteoinductivity of CMC/nHA/GO may profit from the surface chemistry and roughness, which benefit the β1 integrin interactions with the extracellular matrix and activate the FAK–ERK signaling pathway to upregulate the expression of osteogenic special proteins. This study indicates that the nanocomposite scaffold with surface chemistry and roughness modifications could serve as a novel and promising bone substitute for tissue engineering.

The Imbalance of Lymphocyte Subsets and Cytokines: Potential Immunologic Insights Into the Pathogenesis of Chronic Dacryocystitis

Purpose To explore the quantitative distributions of different lymphocyte subsets in the lacrimal sac mucosa and identify changes of Th1- and Th2-associated cytokines in the tears of patients with chronic dacryocystitis. Methods Lacrimal sac mucosal specimens from patients with chronic dacryocystitis were analyzed. Hematoxylin-eosin staining and Masson staining were performed for pathologic analysis, and immunohistochemical staining was performed for the detection of CD3+, CD4+, CD8+, CD20+, Th1, and Th2 lymphocytes. Quantitative real-time PCR was performed to analyze IFN-γ and IL-4 mRNA expression. In addition, tear samples from patients with chronic dacryocystitis and healthy volunteers were collected and analyzed with an antibody array system for Th1- and Th2-associated cytokines and chemokines. Results Different distribution patterns of lymphocyte subsets were observed in the lacrimal sac walls. Both CD20+ B lymphocytes and CD3+ T lymphocytes accumulated in organized lymphoid follicles, and CD3+ T cells were also distributed in a diffuse manner. Among the two subsets of T cells, CD4+ T cells were more abundant than CD8+ T cells. Both the immunohistochemical staining and real-time PCR results revealed significantly higher expression levels of IFN-γ than those of IL-4. The levels of Th1- and Th2-related cytokines and chemokines measured were significantly higher in the tears of patients than in those of controls. Conclusions The different distribution patterns of lymphocyte subsets provide insight into a potential immunologic mechanism for dacryocystitis. The cytokines secreted by Th1 or Th2 cells may play a major role in the pathogenesis of dacryocystitis and could be explored as therapeutic targets.