Enhanced Bone Tissue Regeneration by Porous Gelatin Composites Loaded with the Chinese Herbal Decoction Danggui Buxue Tang

Danggui Buxue Tang (DBT) is a traditional Chinese herbal decoction containing Radix Astragali and Radix Angelicae sinensis. Pharmacological results indicate that DBT can stimulate bone cell proliferation and differentiation. The aim of the study was to investigate the efficacy of adding DBT to bone substitutes on bone regeneration following bone injury. DBT was incorporated into porous composites (GGT) made from genipin-crosslinked gelatin and β-triclacium phosphates as bone substitutes (GGTDBT). The biological response of mouse calvarial bone to these composites was evaluated by in vivo imaging systems (IVIS), micro-computed tomography (micro-CT), and histology analysis. IVIS images revealed a stronger fluorescent signal in GGTDBT-treated defect than in GGT-treated defect at 8 weeks after implantation. Micro-CT analysis demonstrated that the level of repair from week 4 to 8 increased from 42.1% to 71.2% at the sites treated with GGTDBT, while that increased from 33.2% to 54.1% at GGT-treated sites. These findings suggest that the GGTDBT stimulates the innate regenerative capacity of bone, supporting their use in bone tissue regeneration.     

Evaluating the bone regeneration in calvarial defect using osteoblasts differentiated from adipose-derived mesenchymal stem cells on three different scaffolds: an animal study

The aim of this study was to investigate the effect of three different scaffolds on the viability and differentiation of adipose-derived mesenchymal stem cells (ADMSCs) to osteoblast for bone regeneration of calvarial defect in rabbit model. Adipose was harvested from the nape of 12 rabbits by direct surgery or hollow-tip cannula. Two standardized circular calvarial defects (case and control), 8 mm in diameter each, were created in all the animals. The animals were divided into 3 different groups. In group 1 (G1), the defect was filled with polyamide + ADMSC. In group 2, poly lactic-co-glycolic acid + ADMSC was used. In group 3, decellularized amniotic membrane + ADMSC was applied. In the control defect, the non-seeded scaffolds were applied for filling the defect. Decellularized pericardial scaffolds were used as a membrane on the scaffolds. The animals were euthanized 2, 4, and 8 weeks of operation and new bone formation was assessed by different analyses. Immunohistochemical (IHC) staining with osteopontin and osteocalcin antibodies was also performed. After 2 weeks of wound healing, minimal bone regeneration was detected in all groups. Almost complete defect closure was observed in all experimental groups after 8 weeks of operation, with the greatest defect closure in the animals treated with polyamide scaffolds as compared to biopsies obtained from control defects and other experimental groups. The maximal tensile load was higher in G1, 4 and 8 weeks postoperatively, suggesting the usefulness of polyamide + ADMSC for bone regeneration in calvarial defects. Results of the IHC staining demonstrated a significant difference between seeded and non-seeded scaffold in both short- and long-term follow-ups (P < 0.05). In addition, a significant difference was observed in enhancement of IHC staining of both markers in polyamide group (seeded or non-seeded) 4 and 8 weeks postoperatively in comparison with other scaffolds. It was concluded that bone regeneration in critical calvarial defect was more successful in seeded polyamide.     

Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects

Introduction

Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM).

Materials and Methods

The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5).

Results

PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups.

Conclusion

Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus.     

SDF-1 Enhances Wound Healing of Critical-Sized Calvarial Defects beyond Self-Repair Capacity

Host blood circulating stem cells are an important cell source that participates in the repair of damaged tissues. The clinical challenge is how to improve the recruitment of circulating stem cells into the local wound area and enhance tissue regeneration. Stromal-derived factor-1 (SDF-1) has been shown to be a potent chemoattractant of blood circulating stem cells into the local wound microenvironment. In order to investigate effects of SDF-1 on bone development and the repair of a large bone defect beyond host self-repair capacity, the BMP-induced subcutaneous ectopic bone formation and calvarial critical-sized defect murine models were used in this preclinical study. A dose escalation of SDF-1 were loaded into collagen scaffolds containing BMP, VEGF, or PDGF, and implanted into subcutaneous sites at mouse dorsa or calvarial critical-sized bone defects for 2 and 4 weeks. The harvested biopsies were examined by microCT and histology. The results demonstrated that while SDF-1 had no effect in the ectopic bone model in promoting de novo osteogenesis, however, in the orthotopic bone model of the critical-sized defects, SDF-1 enhanced calvarial critical-sized bone defect healing similar to VEGF, and PDGF. These results suggest that SDF-1 plays a role in the repair of large critical-sized defect where more cells are needed while not impacting de novo bone formation, which may be associated with the functions of SDF-1 on circulating stem cell recruitment and angiogenesis.     

Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration

There is a growing need for successful bone tissue engineering strategies and advanced biomaterials that mimic the structure and function of native tissues carry great promise. Successful bone repair approaches may include an osteoconductive scaffold, osteoinductive growth factors, cells with an osteogenic potential and capacity for graft vascularisation. To increase osteoinductivity of biomaterials, the local combination and delivery of growth factors has been developed. In the present study we investigated the osteogenic effects of calcium phosphate (CaP)-coated nanofiber mesh tube-mediated delivery of BMP-7 from a PRP matrix for the regeneration of critical sized segmental bone defects in a small animal model. Bilateral full-thickness diaphyseal segmental defects were created in twelve male Lewis rats and nanofiber mesh tubes were placed around the defect. Defects received either treatment with a CaP-coated nanofiber mesh tube (n?=?6), an un-coated nanofiber mesh tube (n=6) a CaP-coated nanofiber mesh tube with PRP (n=6) or a CaP-coated nanofiber mesh tube in combination with 5?μg?BMP-7 and PRP (n?=?6). After 12?weeks, bone volume and biomechanical properties were evaluated using radiography, microCT, biomechanical testing and histology. The results demonstrated significantly higher biomechanical properties and bone volume for the BMP group compared to the control groups. These results were supported by the histological evaluations, where BMP group showed the highest rate of bone regeneration within the defect. In conclusion, BMP-7 delivery via PRP enhanced functional bone defect regeneration, and together these data support the use of BMP-7 in the treatment of critical sized defects.     

Dynamic Mechanical and Nanofibrous Topological Combinatory Cues Designed for Periodontal Ligament Engineering

Complete reconstruction of damaged periodontal pockets, particularly regeneration of periodontal ligament (PDL) has been a significant challenge in dentistry. Tissue engineering approach utilizing PDL stem cells and scaffolding matrices offers great opportunity to this, and applying physical and mechanical cues mimicking native tissue conditions are of special importance. Here we approach to regenerate periodontal tissues by engineering PDL cells supported on a nanofibrous scaffold under a mechanical-stressed condition. PDL stem cells isolated from rats were seeded on an electrospun polycaprolactone/gelatin directionally-oriented nanofiber membrane and dynamic mechanical stress was applied to the cell/nanofiber construct, providing nanotopological and mechanical combined cues. Cells recognized the nanofiber orientation, aligning in parallel, and the mechanical stress increased the cell alignment. Importantly, the cells cultured on the oriented nanofiber combined with the mechanical stress produced significantly stimulated PDL specific markers, including periostin and tenascin with simultaneous down-regulation of osteogenesis, demonstrating the roles of topological and mechanical cues in altering phenotypic change in PDL cells. Tissue compatibility of the tissue-engineered constructs was confirmed in rat subcutaneous sites. Furthermore, in vivo regeneration of PDL and alveolar bone tissues was examined under the rat premaxillary periodontal defect models. The cell/nanofiber constructs engineered under mechanical stress showed sound integration into tissue defects and the regenerated bone volume and area were significantly improved. This study provides an effective tissue engineering approach for periodontal regeneration—culturing PDL stem cells with combinatory cues of oriented nanotopology and dynamic mechanical stretch.     

In vitro and in vivo evaluation of xenogeneic bone putty with the carrier of hydrogel derived from demineralized bone matrix

The demineralized bone matrix (DBM) putty is a traditional bone graft utilized to facilitate the repair and reconstruction of bone. Recent studies indicated the DBM putties with the various carriers were different in bone repairing ability. In order to prepare a kind of DBM putty with a good biocompatibility and bioactivity, the DBM gel was processed from the DBM and the feasibility as a carrier for the DBM putty was evaluated. After the bovine DBM gel was prepared, the BMPs content as well as the ability to promote osteogenic differentiation of MC3T3-E1 cells in vitro were investigated. Then the DBM putty was prepared and filled into the rat calvarial defect model to evaluate the bone repairing ability by micro-CT and histology. The result showed there was 2.953?±?0.054 ng BMP contained in per gram of the DBM gel. And the ALP production of MC3T3-E1 cells in the DBM gels group increased with prolonged culturing, the mineralized nodules formed in MC3T3-E1 cells on 14th day after co-culture. The putty prepared by DBM gel was easy to handle without loss of DBM particles at room temperature. In the rat calvarial bone defect experiment, histological observation showed more mature bone formed in the DBM putty group than that in the type I collagen group at 12 weeks, which indicated the bone putty prepared by DBM gel exhibited a better bone repair capability.     

Osteogenesis in calvarial defects: contribution of the dura,the pericranium,and the surrounding bone in adult versus infant animals

Guided bone regeneration is a promising means for reconstructing bone defects in the cranium. The present study was performed to better define those factors that affect osteogenesis in the cranium. The authors studied a single animal model, investigating the contribution of the dura, the pericranium, and the adjacent calvarial bone in the process of calvarial regeneration in both mature and immature animals. Bilateral, 100-mm2, parietal calvariectomies were performed in immature (n = 16) and mature (n = 16) rabbits. Parietal defects were randomized to one of four groups depending on the differential blockade of the dura and/or the pericranium by expanded polytetrafluoroethylene membranes. Animals were humanely killed after 12 weeks, and histometric analysis was performed to quantitate the area of the original bone defect, new bone formation, and new bone density. Bone formation was quantified separately both at the periphery and in the center of the defects. Extrasite bone formation was also quantified both on the dural and on the pericranial sides of the barriers. Bone regeneration was incomplete in all groups over the 12-week study period, indicating that complete bone healing was not observed in any group. The dura was more osteogenic than the pericranium in mature and immature animals, as there was significantly more extrasite bone formed on the dural side in the double expanded polytetrafluoroethylene barrier groups. In both the dural and the double expanded polytetrafluoroethylene barrier groups, dural bone production was significantly greater in immature compared with mature animals. The dura appeared to be the source of central new bone, because dural blockade in the dural and double expanded polytetrafluoroethylene groups resulted in a significant decrease in central bone density in both mature and immature animals. Paradoxically, isolation of the pericranium in mature animals resulted in a significant reduction in total new bone area, whereas pericranial contact appeared to enhance peripheral new bone formation, with the control group having the greatest total new bone area. The present study establishes a model to quantitatively study the process of bone regeneration in calvarial defects and highlights differences in the contribution of the dura and pericranium to calvarial bone regeneration between infant and adult animals. On the basis of these findings, the authors propose that subsequent studies in which permeability of the expanded polytetrafluoroethylene membranes is altered to permit migration of osteoinductive proteins into the defect while blocking prolapse of adjacent soft tissues may help to make guided bone regeneration a realistic alternative for the repair of cranial defects.     

Injectable gel with synthetic collagen-binding peptide for enhanced osteogenesis in vitro and in vivo

A synthetic peptide denoted as collagen-binding motif (CBM) was identified from osteopontin (OPN), a multisubunit extracellular matrix (ECM) protein, by enzymatic digestion with chymotrypsin. The aim of this study was to examine the feasibility of identified CBM peptide as an active component of gel type scaffold material in osteogenesis. The binding of CBM peptide to collagen was specific and presented high affinity. Cell adhesion and growth on CBM peptide-immobilized gel were significantly increased as compared with those on gel with control peptide or without peptide. The CBM peptide-immobilized gel increased osteoblastic differentiation, followed by marked bone formation in the rabbit calvarial defect sites at 4 weeks. Taken together, the injectable gel with synthetic CBM peptide has a potential to induce osteogenesis in vitro and in vivo, suggesting its clinical application in bone regeneration procedure.     

Mechanisms and control of silk-based electrospinning

Silk fibroin (SF) nanofibers, formed through electrospinning, have attractive utility in regenerative medicine due to the biocompatibility, mechanical properties, and tailorable degradability. The mechanism of SF electrospun nanofiber formation was studied to gain new insight into the formation and control of nanofibers. SF electrospinning solutions with different nanostructures (nanospheres or nanofilaments) were prepared by controlling the drying process during the preparation of regenerated SF films. Compared to SF nanospheres in solution, SF nanofilaments had better spinnability with lower viscosity when the concentration of silk protein was below 10%, indicating a critical role for SF morphology, and in particular, nanostructures, for the formation of electrospun fibers. More interesting, the diameter of electrospun fibers gradually increased from 50 to 300 nm as the concentration of SF nanofilaments in the solution increased from 6 to 12%, implying size control by simply adjusting SF nanostructure and concentration. Aside from process parameters investigated in previous studies, such as SF concentration, viscosity, and electrical potential, the present mechanism emphasizes significant influence of SF nanostructure on spinnability and diameter control of SF electrospun fibers, providing a controllable option for the preparation of silk-based electrospun scaffolds for biomaterials, drug delivery, and tissue engineering needs.     

Applications of a mouse model of calvarial healing: differences in regenerative abilities of juveniles and adults

Young children are capable of healing large calvarial defects, whereas adults lack this endogenous osseous tissue-engineering capacity. Despite the important clinical implications, little is known about the molecular and cell biology underlying this differential ability. Traditionally, guinea pig, rabbit, and rat models have been used to study the orchestration of calvarial healing. To harness the research potential of knockout and transgenic mice, the authors developed a mouse model for calvarial healing. Nonsuture-associated parietal defects 3, 4, and 5 mm in diameter were made in both juvenile (6-day-old, n = 15) and adult (60-day-old, n = 15) mice. Calvariae were harvested after 8 weeks and analyzed radiographically and histologically. Percentage of healing was quantified using Scion Image software analysis of calvarial radiographs. A significant difference in the ability to heal calvarial defects was seen between 6-day-old and 60-day-old mice when 3-, 4-, or 5-mm defects were created. The authors' analysis revealed that juvenile mice healed a significantly greater percentage of their calvarial defects than adult mice (juvenile mean percentage of healing: 3-mm defects, 59 percent; 4-mm defects, 65 percent; 5-mm defects, 44 percent; adult mean percentage of healing: <5 percent in all groups; p < 0.05). All three defect sizes were found to be critical in the adult, whereas significant healing was seen regardless of the size of the defect in juvenile mice. The establishment of this model will facilitate further, detailed evaluation of the molecular biology underlying the different regenerative abilities of juvenile versus adult mice and enhance research into membranous bone induction by making available powerful tools such as knockout and transgenic animals.     

Human amniotic fluid stem cells attract osteoprogenitor cells in bone healing

Current treatments of large bone defects are based on autologous or allogenic bone transplantation. Human amniotic fluid stem cells (hAFSCs) were evaluated for their potential in bone regenerative medicine. In this study, hAFSCs were transduced with lentiviral vector harboring red fluorescent protein to investigate their role in the regeneration of critical-size bone defects in calvarial mouse model. To distinguish donor versus recipient cells, a transgenic mouse model carrying GFP fluorescent reporter was used as recipient to follow the fate of hAFSCs transplanted in vivo into Healos® scaffold. Our results showed that transduced hAFSCs can be tracked in vivo directly at the site of transplantation. The presence of GFP positive cells in the scaffold at 3 and 6 weeks after transplantation indicates that donor hAFSCs can recruit host cells during the repair process. These observations help clarify the role of hAFSCs in bone tissue repair.     

It is Time for a Change:Petri Dishes Weaken Cells

We wish to draw the attention to a potential deficiency in the biocompatibility of polystyrene cell culturc dishes which is caused by a softening of the material under relevant culture conditions.The finding confirms the central hypothesis of our previous model study.In it we assumed a local increase in pH at the interface between the hydrophilic polymer and liquid.The finding is of considerable biological interest.Polystyrene tissue culture dishes are now in use for 50 years.To the best of our knowledge their biocompatibility has never been challenged.Here we report the first experimental proof that exposure to water softens the surface of polystyrene Petri dishes.We expect that our results will stimulate the development ofa new generation of cell culture devices,including Petri dishes and culture flasks,and the establishment of improved biomimetic settings for tissuc engineering and stem cell research.New non-swelling biomaterials or nanocoatings designed to reduce the swelling of polymer culture dishes could improve cell performance.The need for further study is clear.     

The Angiopoietin-1 Variant COMP-Ang1 Enhances BMP2-Induced Bone Regeneration with Recruiting Pericytes in Critical Sized Calvarial Defects

Craniofacial bone defects are observed in a variety of clinical situations, and their reconstructions require coordinated coupling between angiogenesis and osteogenesis. In this study, we explored the effects of cartilage oligomeric matrix protein-angiopoietin 1 (COMP-Ang1), a synthetic and soluble variant of angiopoietin 1, on bone morphogenetic protein 2 (BMP2)-induced cranial bone regeneration, and recruitment and osteogenic differentiation of perivascular pericytes. A critical-size calvarial defect was created in the C57BL/6 mouse and COMP-Ang1 and/or BMP2 proteins were delivered into the defects with absorbable collagen sponges. After 3 weeks, bone regeneration was evaluated using micro-computed tomography and histologic examination. Pericyte recruitment into the defects was examined using immunofluorescence staining with anti-NG2 and anti-CD31 antibodies. In vitro recruitment and osteoblastic differentiation of pericyte cells were assessed with Boyden chamber assay, staining of calcified nodules, RT-PCR and Western blot analyses. Combined administration of COMP-Ang1 and BMP2 synergistically enhanced bone repair along with the increased population of CD31 (an endothelial cell marker) and NG2 (a specific marker of pericyte) positive cells. In vitro cultures of pericytes consistently showed that pericyte infiltration into the membrane pore of Boyden chamber was more enhanced by the combination treatment. In addition, the combination further increased the osteoblast-specific gene expression, including bone sialoprotein (BSP), osteocalcin (OCN) and osterix (OSX), phosphorylation of Smad/1/5/8, and mineralized nodule formation. COMP-Ang1 can enhance BMP2-induced cranial bone regeneration with increased pericyte recruitment. Combined delivery of the proteins might be a therapeutic strategy to repair cranial bone damage.     

Biocompatibility of nano-hydroxyapatite/Mg-Zn-Ca alloy composite scaffolds to human umbilical cord mesenchymal stem cells from Wharton’s jelly in vitro

Seeding cells and scaffolds play pivotal roles in bone tissue engineering and regenerative medicine.Wharton’s jelly-derived mesenchymal stem cells(WJCs)from human umbilical cord represent attractive and promising seeding cells in tissue regeneration and engineering for treatment applications.This study was carried out to explore the biocompatibility of scaffolds to seeding cells in vitro.Rod-like nano-hydroxyapatite(RN-HA)and flake-like micro-hydroxyapatite(FM-HA)coatings were prepared on Mg-Zn-Ca alloy substrates using micro-arc oxidation and electrochemical deposition.WJCs were utilized to investigate the cellular biocompatibility of Mg-Zn-Ca alloys after different surface modifications by observing the cell adhesion,morphology,proliferation,and osteoblastic differentiation.The in vitro results indicated that the RN-HA coating group was more suitable for cell proliferation and cell osteoblastic differentiation than the FM-HA group,demonstrating better biocompatibility.Our results suggested that the RN-HA coating on Mg-Zn-Ca alloy substrates might be of great potential in bone tissue engineering.     

Evaluation of processed bovine cancellous bone matrix seeded with syngenic osteoblasts in a critical size calvarial defect rat model

INTRODUCTION: Biologic bone substitutes may offer alternatives to bone grafting procedures. The aim of this study was to evaluate a preformed bone substitute based on processed bovine cancellous bone (PBCB) with or without osteogenic cells in a critical size calvarial defect rat model. METHODS: Discs of PBCB (Tutobone) were seeded with second passage fibrin gel-immobilized syngenic osteoblasts (group A, n = 40). Cell-free matrices (group B, n = 28) and untreated defects (group C; n=28) served as controls. Specimens were explanted between day 0 and 4 months after implantation and were subjected to histological and morphometric evaluation. RESULTS: At 1 month, bone formation was limited to small peripheral areas. At 2 and 4 months, significant bone formation, matrix resorption as well as integration of the implants was evident in groups A and B. In group C no significant regeneration of the defects was observed. Morphometric analysis did not disclose differences in bone formation in matrices from groups A and B. Carboxyfluorescine-Diacetate-Succinimidylester (CFDA) labeling demonstrated low survival rates of transplanted cells. DISCUSSION: Osteoblasts seeded into PBCB matrix display a differentiated phenotype following a 14 days cell culture period. Lack of initial vascularization may explain the absence of added osteogenicity in constructs from group A in comparison to group B. PBCB is well integrated and represents even without osteogenic cells a promising biomaterial for reconstruction of critical size calvarial bone defects.     

Dexamethasone-loaded hydroxyapatite enhances bone regeneration in rat calvarial defects

A combination of bioceramics and osteogenic factors is potentially useful for bone regeneration applications. In the present study, hydroxyapatite particles (HA) were loaded with dexamethasone (Dex) and then characterized using SEM and drug release study. The bone regeneration ability of Dex-loaded HA (Dex/HA) was investigated in a rat critical size bone defect using digital mammography, multislice spiral-computed tomography (MSCT) imaging, and histological analysis. The HA and Dex/HA showed nano and micro-scale morphology with a nearly homogenous distribution of diameter. In addition, about 90 % of the drug was released from Dex/HA over a period of three days. After 8 weeks of implantation in rat calvarial defects, no sign of inflammation or complication was observed at the site of surgery. According to digital mammography and MSCT, Dex/HA showed the highest bone regeneration in rat bone defects compared to those received drug-free HA. Histological studies confirmed these data and showed osteointegration to the surrounding tissue. Taking all together, it was demonstrated that Dex/HA can be used as an appropriate synthetic graft for bone tissue engineering applications. These newly developed bioceramics can be used as new bone graft substitutes in orthopaedic surgery and is capable of enhancing bone regeneration.     

A Silk Fibroin/Collagen Nerve Scaffold Seeded with a Co-Culture of Schwann Cells and Adipose-Derived Stem Cells for Sciatic Nerve Regeneration

As a promising alternative to autologous nerve grafts, tissue-engineered nerve grafts have been extensively studied as a way to bridge peripheral nerve defects and guide nerve regeneration. The main difference between autogenous nerve grafts and tissue-engineered nerve grafts is the regenerative microenvironment formed by the grafts. If an appropriate regenerative microenvironment is provided, the repair of a peripheral nerve is feasible. In this study, to mimic the body’s natural regenerative microenvironment closely, we co-cultured Schwann cells (SCs) and adipose-derived stem cells (ADSCs) as seed cells and introduced them into a silk fibroin (SF)/collagen scaffold to construct a tissue-engineered nerve conduit (TENC). Twelve weeks after the three different grafts (plain SF/collagen scaffold, TENC, and autograft) were transplanted to bridge 1-cm long sciatic nerve defects in rats, a series of electrophysiological examinations and morphological analyses were performed to evaluate the effect of the tissue-engineered nerve grafts on peripheral nerve regeneration. The regenerative outcomes showed that the effect of treatment with TENCs was similar to that with autologous nerve grafts but superior to that with plain SF/collagen scaffolds. Meanwhile, no experimental animals had inflammation around the grafts. Based on this evidence, our findings suggest that the TENC we developed could improve the regenerative microenvironment and accelerate nerve regeneration compared to plain SF/collagen and may serve as a promising strategy for peripheral nerve repair.     

Osteogenesis in cranial defects: reassessment of the concept of critical size and the expression of TGF-beta isoforms

Transforming growth factor-betas (TGF-beta) have been demontstrated to be upregulated during osteoblast function in vitro and during cranial suture fusion in vivo. The authors hypothesized that spontaneous reossification of calvarial defects was also associated with upregulation of TGF-beta. The present study was designed to (1) evaluate the concept of a critical-size defect within the calvaria in an adult guinea pig model and (2) investigate the association between the ossification of calvarial defects and TGF-beta upregulation. Paired circular parietal defects with diameters of 3 and 5 mm and single parietal defects with diameters of 8 or 12 mm were made in 45 six-month-old skeletally mature guinea pigs. Three animals per defect size were killed after survival periods of 3 days, 1 week, 4 weeks, 8 weeks, or 12 weeks. New bone ingrowth was evaluated by assessing for linear closure by a traditional linear method and by a modified cross-sectional area method using an image analysis system in which the thickness of new bone was taken into account. Immunohistochemistry was performed using rabbit polyclonal antibodies to localize TGF-beta1, -beta2, and -beta3. All specimens were photographed, and the intensity of immunostaining was graded based on subjective photographic assessment by three independent reviewers. No defect demonstrated any measurable bone replacement after a survival period of 3 days. All 3- and 5-mm defects were completely reossified after 12 weeks based on the linear analysis of new bone, indicating these defects to be less than critical size. However, new bone formation in the 5-mm defects never exceeded a mean of 40 percent by cross-sectional area of new bone. Percent of new bone formation by cross-sectional area was significantly higher within 3-mm defects than in all larger defects 4 weeks after the craniotomy, reaching a mean of 89 percent new bone by 12 weeks. Persistent gaps were noted on linear analysis of the 8- and 12-mm wounds by 12 weeks, and mean percent new bone by cross-sectional area remained below 30 percent. Immunolocalization demonstrated osteogenic fronts at the advancing bone edge and the endocranial side, in which the osteoblasts stained strongly for all isoforms of TGF-beta. The intensity of osteoblast expression waned considerably after the majority of the defect had reossified. These data indicate that histometric analysis based on cross-sectional area more accurately reflects the osteogenic potential of a cranial defect than does linear inspection of defect closure. Although the interpretation of immunolocalization studies is highly subjective, independent assessment by three reviewers indicates that isoforms of TGF-beta were upregulated during a limited "window" of time corresponding to the period of active calvarial reossification, and expression of TGF-beta corresponded to osteoblast activity within osteogenic fronts.