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Small bone defects can heal spontaneously through the bone modeling process due to their physiological environmental conditions. The bone modeling cycle preserves the reliability of the skeleton through the well-adjusted activities of its fundamental cell. Stem cells are a source of pluripotent cells with a capacity to differentiate into any tissue in the existence of a suitable medium. The concept of bone engineering is based on stem cells that can differentiate into bone cells. Mesenchymal stromal cells have been evaluated in bone tissue engineering due to their capacity to differentiate in osteoblasts. They can be isolated from bone marrow and from several adults oral and dental tissues such as permanent or deciduous teeth dental pulp, periodontal ligament, apical dental papilla, dental follicle precursor cells usually isolated from the follicle surrounding the third molar, gingival tissue, periosteum-derived cells, dental alveolar socket, and maxillary sinus Schneiderian membrane-derived cells. Therefore, a suitable animal model is a crucial step, as preclinical trials, to study the outcomes of mesenchymal cells on the healing of bone defects. We will discuss, through this paper, the use of mesenchymal stem cells obtained from several oral tissues mixed with different types of scaffolds tested in different animal models for bone tissue engineering. We will explore and link the comparisons between human and animal models and emphasized the factors that we need to take into consideration when choosing animals. The pig is considered as the animal of choice when testing large size and multiple defects for bone tissue engineering., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The repair of bone defects is a big challenge in reconstructive surgery. Periosteal distraction osteogenesis (PDO), as a promising technique used for bone regeneration, forms a space between the periosteum and bone cortex to regenerate the new bone merely by distracting the periosteum. In order to investigate the influence of distractor framework on the PDO, we utilized three-dimensional (3D) printing technology to fabricate three kinds of poly-L-lactic acid (PLLA) scaffolds with different pore sizes in this study. The in vitro experiments showed that the customized PLLA scaffolds had different-sized microchannels with low toxicity, good biocompatibility, and enough mechanical strength. Then, we built up an in vivo bioreactor under the skull periosteum of New Zealand white rabbits. The distractors with different pore sizes all could satisfy the demand of periosteal distraction in the animal experiments. After 8 weeks of consolidation period, the quality and quantity of the newly formed bone were improved with the increasing pore sizes of the distractors. Moreover, the newly formed bone also displayed an increasing degree of vascularization. In conclusion, 3D printing technology could promote the innovation of PDO devices and fabricate optimized scaffolds with appropriate pore sizes, shapes, and structures. It would help us regenerate more functional tissue-engineered bone and provide new ideas for further clinical application of the PDO technique., Competing Interests: The authors declared that they have no conflicts of interest., (Copyright © 2020 Danyang Zhao et al.)

An attractive alternative to bone autografts is the use of autologous mesenchymal progenitor cells (MSCs) in combination with biomaterials. We compared the therapeutic potential of different sources of mesenchymal stem cells in combination with biomaterials in a bone nonunion model. A critical-size defect was created in Sprague-Dawley rats. Animals were divided into six groups, depending on the treatment to be applied: bone defect was left empty (CTL); treated with live bone allograft (LBA); hrBMP-2 in collagen scaffold (CS BMP2 ); acellular polycaprolactone scaffold (PCL group); PCL scaffold containing periosteum-derived MSCs (PCL PMSCs ) and PCL containing bone marrow-derived MSCs (PCL BMSCs ). To facilitate cell tracking, both MSCs and bone graft were isolated from green fluorescent protein (GFP)-transgenic rats. CTL group did not show any signs of healing during the radiological follow-up (n = 6). In the LBA group, all the animals showed bone bridging (n = 6) whereas in the CS BMP2 group, four out of six animals demonstrated healing. In PCL and PCL PMSCs groups, a reduced number of animals showed radiological healing, whereas no healing was detected in the PCL BMSCs group. Using microcomputed tomography, the bone volume filling the defect was quantified, showing significant new bone formation in the LBA, CS BMP2 , and PCL PMSCs groups when compared with the CTL group. At 10 weeks, GFP positive cells were detected only in the LBA group and restricted to the outer cortical bone in close contact with the periosteum. Tracking of cellular implants demonstrated significant survival of the PMSCs when compared with BMSCs. In conclusion, PMSCs improve bone regeneration being suitable for mimetic autograft design., (© 2019 John Wiley & Sons, Ltd.)

Large mandibular defects are clinically challenging to reconstruct due to the complex anatomy of the jaw and the limited availability of appropriate tissue for repair. We envision leveraging current advances in fabrication and biomaterials to create implantable devices that generate bone within the patients themselves suitable for their own specific anatomical pathology. The in vivo bioreactor strategy facilitates the generation of large autologous vascularized bony tissue of customized geometry without the addition of exogenous growth factors or cells. To translate this technology, we investigated its success in reconstructing a mandibular defect of physiologically relevant size in sheep. We fabricated and implanted 3D-printed in vivo bioreactors against rib periosteum and utilized biomaterial-based space maintenance to preserve the native anatomical mandibular structure in the defect site before reconstruction. Nine weeks after bioreactor implantation, the ovine mandibles were repaired with the autologous bony tissue generated from the in vivo bioreactors. We evaluated tissues generated in bioreactors by radiographic, histological, mechanical, and biomolecular assays and repaired mandibles by radiographic and histological assays. Biomaterial-aided mandibular reconstruction was successful in a large superior marginal defect in five of six (83%) sheep. Given that these studies utilized clinically available biomaterials, such as bone cement and ceramic particles, this strategy is designed for rapid human translation to improve outcomes in patients with large mandibular defects., Competing Interests: The authors declare no conflict of interest.

Periosteal osteogenic capacity can be exploited to enhance bone formation in the fields of tissue engineering and regenerative medicine. Despite this importance, there have been no studies examining the composition, structure, and osteogenic capacity of periostea from different bone sources. In this study, structure and osteogenic factor content were compared among periostea from rib, calvarial, femoral, and tibial bones, in which the native bones of these four regions were harvested and subjected to histological analysis. The osteogenic capacity of grafted periosteum was evaluated using an in vivo vascularized pedicle model of bone tissue engineering. Poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogels were seeded with bone marrow mesenchymal stem cells and implanted with grafted periosteum harvested from either calvarial or tibial bone, which were representative of thin and thick native periostea, respectively. The cambium layer thickness of periostea from the femoral and tibial bones (36.9% ± 2.5% and 36.8% ± 2.6%) was greater than that from the calvarial and rib bones (26.8% ± 2.4% and 25.5% ± 1.9%). The osteocalcin and alkaline phosphatase levels were comparatively higher in the femoral and tibial periostea than those in periostea harvested from the calvarial and rib bones. The construct implanted with grafted tibial periosteum resulted in greater neo-bone regeneration and higher osteocalcin and alkaline phosphatase expression. This study is the first investigation of the osteogenic capacity of periostea from diverse sources. The results can be used to guide clinical strategies that exploit periostea for tissue engineering and clinical applications.

Background: Chondrogenic mesenchymal stem cells (MSCs) have not yet been used to address the clinical demands of large osteochondral joint surface defects. In this study, self-assembling tissue intermediates (TIs) derived from human periosteum-derived stem/progenitor cells (hPDCs) were generated and validated for stable cartilage formation in vivo using two different animal models., Methods: hPDCs were aggregated and cultured in the presence of a novel growth factor (GF) cocktail comprising of transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP)2, growth differentiation factor (GDF)5, BMP6, and fibroblast growth factor (FGF)2. Quantitative polymerase chain reaction (PCR) and immunohistochemistry were used to study in vitro differentiation. Aggregates were then implanted ectopically in nude mice and orthotopically in critical-size osteochondral defects in nude rats and evaluated by microcomputed tomography (µCT) and immunohistochemistry., Results: Gene expression analysis after 28 days of in vitro culture revealed the expression of early and late chondrogenic markers and a significant upregulation of NOGGIN as compared to human articular chondrocytes (hACs). Histological examination revealed a bilayered structure comprising of chondrocytes at different stages of maturity. Ectopically, TIs generated both bone and mineralized cartilage at 8 weeks after implantation. Osteochondral defects treated with TIs displayed glycosaminoglycan (GAG) production, type-II collagen, and lubricin expression. Immunostaining for human nuclei protein suggested that hPDCs contributed to both subchondral bone and articular cartilage repair., Conclusion: Our data indicate that in vitro derived osteochondral-like tissues can be generated from hPDCs, which are capable of producing bone and cartilage ectopically and behave orthotopically as osteochondral units.

The repair of large bone defects with complex geometries remains a major clinical challenge. Here, we explored the feasibility of fabricating polylactic acid-hydroxyapatite (PLA-HA) composite scaffolds. These scaffolds were constructed from vascularized tissue engineered bone using an in vivo bioreactor (IVB) strategy with three-dimensional printing technology. Specifically, a rabbit model was established to prefabricate vascularized tissue engineered bone in two groups. An experimental group (EG) was designed using a tibial periosteum capsule filled with 3D printed (3DP) PLA-HA composite scaffolds seeded with bone marrow stromal cells (BMSCs) and crossed with a vascular bundle. 3DP PLA-HA scaffolds were also combined with autologous BMSCs and transplanted to tibial periosteum without blood vessel as a control group (CG). After four and eight weeks, neovascularisation and bone tissues were analysed by studying related genes, micro-computed tomography (Micro-CT) and histological examinations between groups. The results showed that our method capably generated vascularized tissue engineered bone in vivo. Furthermore, we observed significant differences in neovascular and new viable bone formation in the two groups. In this study, we demonstrated the feasibility of generating large vascularized bone tissues in vivo with 3DP PLA-HA composite scaffolds.

A principal purpose of tissue engineering is the augmentation, repair or replacement of diseased or injured human tissue. This study was undertaken to determine whether human biopsies as a cell source could be utilized for successful engineering of human phalanges consisting of both bone and cartilage. This paper reports the use of cadaveric human chondrocytes and periosteum as a model for the development of phalanx constructs. Two factors, osteogenic protein-1 [OP-1/bone morphogenetic protein-7 (BMP7)], alone or combined with insulin-like growth factor (IGF-1), were examined for their potential enhancement of chondrocytes and their secreted extracellular matrices. Design of the study included culture of chondrocytes and periosteum on biodegradable polyglycolic acid (PGA) and poly-l-lactic acid (PLLA)-poly-ε-caprolactone (PCL) scaffolds and subsequent implantation in athymic nu/nu (nude) mice for 5, 20, 40 and 60 weeks. Engineered constructs retrieved from mice were characterized with regard to genotype and phenotype as a function of developmental (implantation) time. Assessments included gross observation, X-ray radiography or microcomputed tomography, histology and gene expression. The resulting data showed that human cell-scaffold constructs could be successfully developed over 60 weeks, despite variability in donor age. Cartilage formation of the distal phalanx models enhanced with both OP-1 and IGF-1 yielded more cells and extracellular matrix (collagen and proteoglycans) than control chondrocytes without added factors. Summary data demonstrated that human distal phalanx models utilizing cadaveric chondrocytes and periosteum were successfully fabricated and OP-1 and OP-1/IGF-1 accelerated construct development and mineralization. The results suggest that similar engineering and transplantation of human autologous tissues in patients are clinically feasible. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Successful application of cell-based strategies in cartilage and bone tissue engineering has been hampered by the lack of robust protocols to efficiently differentiate mesenchymal stem cells into the chondrogenic lineage. The development of chemically defined culture media supplemented with growth factors (GFs) has been proposed as a way to overcome this limitation. In this work, we applied a fractional design of experiment (DoE) strategy to screen the effect of multiple GFs (BMP2, BMP6, GDF5, TGF-β1, and FGF2) on chondrogenic differentiation of human periosteum-derived mesenchymal stem cells (hPDCs) in vitro. In a micromass culture (μMass) system, BMP2 had a positive effect on glycosaminoglycan deposition at day 7 (p < 0.001), which in combination with BMP6 synergistically enhanced cartilage-like tissue formation that displayed in vitro mineralization capacity at day 14 (p < 0.001). Gene expression of μMasses cultured for 7 days with a medium formulation supplemented with 100 ng/mL of BMP2 and BMP6 and a low concentration of GDF5, TGF-β1, and FGF2 showed increased expression of Sox9 (1.7-fold) and the matrix molecules aggrecan (7-fold increase) and COL2A1 (40-fold increase) compared to nonstimulated control μMasses. The DoE analysis indicated that in GF combinations, BMP2 was the strongest effector for chondrogenic differentiation of hPDCs. When transplanted ectopically in nude mice, the in vitro-differentiated μMasses showed maintenance of the cartilaginous phenotype after 4 weeks in vivo. This study indicates the power of using the DoE approach for the creation of new medium formulations for skeletal tissue engineering approaches.

When combining osteogenic progenitor cells such as human periosteum derived cells (hPDCs) with osteoconductive biomaterials like calcium phosphate (CaP)-scaffolds, in vivo bone formation can be achieved. This process is dependent on the early activation of Bone morphogenetic protein (BMP)-signalling. However, the bone forming process is slow and routinely only a limited amount of bone and bone marrow is formed. Therefore, we hypothesised that a robust clinically relevant outcome could be achieved by adding more physiological levels of potent BMP-ligands to these cell- and CaP-based constructs. For this, hPDCs were characterised for their responsiveness to BMP-ligands upon in vitro 2D stimulation. BMP-2, -4, -6 and -9 robustly induced osteochondrogenic differentiation. Subsequently, these ligands were coated onto clinically approved CaP-scaffolds, BioOss® and CopiOs®, followed by hPDC-seeding. Protein lysates and conditioned media were investigated for activation of BMP signalling pathways. Upon in vivo implantation, the most abundant bone formation was found in BMP-2 and BMP-6-coated scaffolds. Implanted cells actively contributed to the newly formed bone. Remnants of cartilage could be observed in BMP-coated CopiOs®-constructs. Computational analysis displayed that the type of BMP-ligand as well as the CaP-scaffold affects skeletal tissue formation, observed in a qualitative as well as quantitative manner. Furthermore, the in vitro mechanism appears to predict the in vivo outcome. This study presents further evidence for the potential of BMP-technology in the development of clinically relevant cell-based constructs for bone regenerative strategies.

The preservation of the bone-forming potential of skeletal progenitor cells during their ex vivo expansion remains one of the major challenges for cell-based bone regeneration strategies. We report that expansion of murine periosteal cells in the presence of FGF2, a signal present during the early stages of fracture healing, is necessary and sufficient to maintain their ability to organize in vivo into a cartilage template which gives rise to mature bone. Implantation of FGF2-primed cells in a large bone defect in mice resulted in complete healing, demonstrating the feasibility of using this approach for bone tissue engineering purposes. Mechanistically, the enhanced endochondral ossification potential of FGF2-expanded periosteal cells is predominantly driven by an increased production of BMP2 and is additionally linked to an improved preservation of skeletal progenitor cells in the cultures. This characteristic is unique for periosteal cells, as FGF2-primed bone marrow stromal cells formed significantly less bone and progressed exclusively through the intramembranous pathway, revealing essential differences between both cell pools. Taken together, our findings provide insight in the molecular regulation of fracture repair by identifying a unique interaction between periosteal cells and FGF2. These insights may promote the development of cell-based therapeutic strategies for bone regeneration which are independent of the in vivo use of growth factors, thus limiting undesired side effects., (© 2014 AlphaMed Press.)

The incorporation of Quality-by-Design (QbD) principles in tissue-engineering bioprocess development toward clinical use will ensure that manufactured constructs possess prerequisite quality characteristics addressing emerging regulatory requirements and ensuring the functional in vivo behavior. In this work, the QbD principles were applied on a manufacturing process step for the in vitro production of osteogenic three-dimensional (3D) hybrid scaffolds that involves cell matrix deposition on a 3D titanium (Ti) alloy scaffold. An osteogenic cell source (human periosteum-derived cells) cultured in a bioinstructive medium was used to functionalize regular Ti scaffolds in a perfusion bioreactor, resulting in an osteogenic hybrid carrier. A two-level three-factor fractional factorial design of experiments was employed to explore a range of production-relevant process conditions by simultaneously changing value levels of the following parameters: flow rate (0.5-2 mL/min), cell culture duration (7-21 days), and cell-seeding density (1.5×10(3)-3×10(3) cells/cm(2)). This approach allowed to evaluate the individual impact of the aforementioned process parameters upon key quality attributes of the produced hybrids, such as collagen production, mineralization level, and cell number. The use of a fractional factorial design approach helped create a design space in which hybrid scaffolds of predefined quality attributes may be robustly manufactured while minimizing the number of required experiments.

The aim of this study was to investigate physical and biological properties of collagen (COL) and demineralized bone powder (DBP) scaffolds for bone tissue engineering. DBP was prepared and divided into three groups, based on various particle sizes: 75-125 µm, 125-250 µm, and 250-500 µm. DBP was homogeneously mixed with type I collagen and three-dimensional scaffolds were constructed, applying chemical crosslinking and lyophilization. Upon culture with human periosteum-derived cells (PD cells), osteogenic differentiation of PD cells was investigated using alkaline phosphatase (ALP) activity and calcium assay kits. The physical properties of the COL/DBP scaffolds were obviously different from COL scaffolds, irrespective of the size of DBP. In addition, PD cells cultured with COL scaffolds showed significantly higher cell adhesion and proliferation than those with COL/DBP scaffolds. In contrast, COL/DBP scaffolds exhibited greater osteoinductive potential than COL scaffolds. The PD cells with COL/DBP scaffolds possessed higher ALP activity than those with COL scaffolds. PD cells cultured with COL/DBP scaffolds with 250-500 mm particle size yielded the maximum calcium deposition. In conclusion, PD cells cultured on the scaffolds could exhibit osteoinductive potential. The composite scaffold of COL/DBP with 250-500 mm particle size could be considered a potential bone tissue engineering implant.

Mesenchymal stem cells (MSCs) isolated from bone marrow and periosteum are often used as cellular sources for bone tissue engineering. This study showed that co-cultured human bone marrow stem cells (hBMSCs) and periosteal-derived stem cells (hPCs) resulted in a synergistic effect on osteogenic differentiation both in vitro and in vivo. Compared to hBMSCs and hPCs, co-culturing MSCs showed abundant mineralization, robust calcium deposition, steadily increasing ALP activity, and upgraded mRNA expression of osteogenic specific genes (COL1A1, BMP-2, osteopontin, osteocalcin) in vitro. Eight weeks after implantation of cellular β-TCP scaffolds in immunodeficient mice, similar synergistic effects were confirmed during in vivo evaluation of total new bone formation, mature bone formation, and neovascularization. Based on these findings, the use of co-cultured hBMSCs and hPCs can be recommended as a promising new approach for bone tissue engineering applications., (Copyright © 2011 John Wiley & Sons, Ltd.)

Background: Periosteal cells are important in embryogenesis, fracture healing, and cartilage repair and could provide cells for osteochondral tissue engineering., Questions/purpose: We determined whether a population of cells isolated from human periosteal tissue contains cells with a mesenchymal stem cell (MSC) phenotype and whether these cells can be expanded in culture and used to form tissue in vitro., Methods: We obtained periosteal tissue from six patients. Initial expression of cell surface markers was assessed using flow cytometry. Cells were cultured over 10 generations and changes in gene expression evaluated to assess phenotypic stability. Phenotype was confirmed using flow cytometry and colony-forming ability assays. Mineral formation was assessed by culturing Stro-1(-) and unsorted cells with osteogenic supplements. Three cell culture samples were used for a reverse transcription-polymerase chain reaction, four for flow cytometry, three for colony-forming assay, and three for mineralization., Results: Primary cultures, containing large numbers of hematopoietic cells were replaced initially by Stro-1 and ALP-expressing immature osteoblastic cell types and later by ALP-expressing cells, which lacked Stro-1 and which became the predominant cell population during subculture. Approximately 10% of the total cell population continued to express markers for Stro1(+)/ALP(-) cells throughout., Conclusions: These data suggest periosteum contains a large number of undifferentiated cells that can differentiate into neotissue and persist despite culture in noncell-specific media for over 10 passages., Clinical Relevance: Cultured periosteal cells may contribute to tissue formation and may be applicable for tissue engineering applications.

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., (© 2011 John Wiley & Sons A/S.)

Background: The aim of this study is to investigate the potential use of periosteum-derived cells (PCs) for tissue engineering in peri-implant defects., Methods: Bone marrow cells (BMCs) and PCs were harvested from seven adult beagle dogs, cultured in vitro, and phenotypically characterized with regard to their osteogenic properties. The animals were then subjected to teeth extraction, and 3 months later, two implant sites were drilled, bone dehiscences created, and dental implants placed. Dehiscences were randomly assigned to one of two groups: PCs (PCs + carrier) and BMCs (BMCs + carrier). After 3 months, the animals were sacrificed and the implants with adjacent hard tissues were processed for undecalcified sections. Bone-to-implant contact, bone fill within the limits of implant threads, and new bone area in a zone lateral to the implant were histometrically obtained., Results: In vitro, phenotypic characterization demonstrated that both cell populations presented osteogenic potential, as identified by the mineral nodule formation and the expression of bone markers. Histometrically, an intergroup analysis showed that both cell-treated defects had similar bone fill within the limits of implant threads and bone-to-implant contact (P >0.05), and although a trend toward higher new bone area values was found for the PC group, there was no significant difference between the experimental groups (P >0.05)., Conclusions: Periosteal and bone marrow cells presented a similar potential for bone reconstruction. As such, periosteum may be considered as an alternative source of osteogenic cells in implant dentistry.

The aim of this study was to establish the potential of human periosteum-derived cells from elderly patients as a cell source for cartilage tissue engineering by optimizing culture conditions for both proliferation and differentiation. Periosteum was obtained from the tibiae of nine patients. Biopsies were prepared for routine histological examination. Periosteum-derived cells were allowed to grow out from the remaining tissue, and were expanded in minimum essential medium containing D-valine (MEM-DV). Fetal bovine serum (FBS) or substitutes, fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1) and non-essential amino acids were added to study proliferation. For differentiation of cells, serum-free medium was used supplemented with one or more isoforms of transforming growth factor-beta (TGFbeta) and/or IGF-1. Samples were analysed for expression of collagens type I, II and X by competitive RT-PCR, immunohistochemically, and histologically using Alcian blue staining. In all samples the cambium layer could hardly be detected. Periosteum-derived cells proliferated in serum-containing MEM-DV. Optimal proliferation was found when this medium was supplemented with 100 ng/ml FGF-2 and non-essential amino acids. Chondrogenesis was detected in 59% of micromasses that were cultured with TGFbeta isomers, and in 83% of the samples cultured in media to which two TGFbeta isoforms were added. Periosteum from elderly humans (mean age 66, range 41-76 years) has chondrogenic potential and remains an attractive cell source for cartilage tissue engineering. By expanding cells in MEM-DV, the selection of progenitor cells might be favoured, which would result in a higher cartilage yield for tissue engineering applications., ((c) 2008 John Wiley & Sons, Ltd.)

Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering., (Copyright 2007 Wiley Periodicals, Inc.)

The purpose of this study was to examine the expression of vascular endothelial growth factor (VEGF) during osteoblastic differentiation of cultured human periosteal-derived cells. Periosteal tissues were obtained from mandible during surgical extraction of lower impacted third molars. Periosteal-derived cells were introduced into cell culture. After passage 3, the periosteal-derived cells were further cultured for 42 days in an osteogenic-inductive culture medium containing dexamethasone, ascorbic acid, and beta-glycerophosphate. The alkaline phosphatase activity in the cultured periosteal-derived cells increased rapidly up to day 14, followed by decrease in activity. The Runx2 protein was expressed at day 7 and day 14, and its expression was not observed thereafter. Both VEGF(165) and VEGF(121) were expressed strongly at days 35 and 42 of culture, particularly during the later stages of differentiation. Alizarin red S-positive nodules were first observed on day 14 and then increased in number during the entire culture period. Osteocalcin and VEGF were first detected in the culture medium on day 14, and their levels increased thereafter in a time-dependent manner. These results suggest that VEGF secretion from cultured human periosteal-derived cells increases along with the mineralization process of the extracellular matrix.

Purpose: To engineer living bone tissue in vitro, bone cells must be multiplied and differentiated in cell culture. Osteoblasts are known to be the crucial cells responsible for the bone modeling process. Periosteal-derived osteoblasts were therefore cultured for up to 3 weeks in Petri dishes as well as in a 3-dimensional collagen gel., Methods: Proliferation, migration, and differentiation of cells as well as the synthesis of extracellular matrix proteins were monitored during the culture period by histology, electron microscopy, and immunohistochemistry. Mineral formation was investigated by electron diffraction studies and element analysis., Results: Osteoblasts proliferated and migrated in Petri dishes as well as in the collagen gel without loss of viability during the whole experimental period. They demonstrated a mature osteoblast phenotype as indicated by the synthesis of a bone-like extracellular matrix. They formed an extracellular matrix containing osteocalcin, osteonectin, and newly synthesized collagen type I in both environments. Mineral formation was seen in colocalization with the bone-like extracellular matrix proteins in Petri dishes. Microanalytical investigations revealed a matrix vesicle-mediated mineral formation at early stages of culture., Conclusions: Our cell culture confirmed the ability to multiplicate differentiated and viable osteoblast-like cells in 2- and 3-dimensional space. Additionally, bone-like mineralization can be induced by primary osteoblasts in monolayer culture. The data suggest that this approach can be used as a tool in bone tissue engineering.

Changes in optical attenuation, relevant to cytochrome oxidase, of the rat bone periosteal tissue in explanted culture and human neuronal cells in three-dimensional agarose constructs have been monitored by the use of optical coherence tomography (OCT), with potential applications in tissue engineering and diagnosis. A superluminescent diode (SLD) with a peak emission wavelength (lambda = 820 nm) that is the near-infrared absorption band of the oxidized form of CytOx was employed. The attenuation coefficient was obtained from the depth-resolved reflectance profiles of liquid phantoms (naphthol green B with intralipid), explant culture (periosteum of calvaria from rats) and cells in 3D agarose constructs. The absorption coefficient of naphthol green B can be accurately quantified by the linear relationship between attenuation coefficients and the concentration. The difference in the attenuation coefficient of astrocytoma cells in agarose before and after reduction of CytOx is 0.26 +/- 0.10 mm(-1) ( n = 9), whereas no attenuation is observed with the agarose control. Reduction of the enzyme in periosteal tissue leads to a change in attenuation coefficient of 0.43 +/- 0.24 mm(-1) ( n = 7). For comparison, using a biochemical assay, the absorption coefficient of the oxidized-reduced form of CytOx is measured at approximately 8.3 +/- 1.5x10(-3) mm-1 ( n = 4) and 8.7 +/-2.5x10(-3) mm-1 ( n = 4) at 820 nm for astrocytoma cells and rat periosteum, respectively. The lower value of CytOx concentration using biochemical versus OCT measurements may result from shifts in the scattering profile and the amplifying influences of multiple heme-based oxidases, indicating that conventional OCT is not specific enough to monitor redox changes in cytochrome oxidase. However, qualitative shifts in oxidation state are apparent using the technique. Our results suggest the potential application of OCT in providing high-resolution tomographic imaging of tissues in organ culture and cells grown in three-dimensional constructs in vitro.

The logical assembly of tissue-engineered bone is ultimately directed by the clinical status of the patient. The basic elements for tissue-engineered bone should include signaling molecules, cells, and extracellular matrix. The assembly of these basic elements may need to be modified by tissue engineers to account for patient variables of age, gender, health, systemic conditions, habits, and anatomical implant. Moreover, different regions of the body will have different functional loads and vascularity. This review discusses several basic options that may be necessary to engineer bone, including spatial and temporal assembly of signaling factors, cells, and biomimetic extracellular matrices. Moreover, the importance of the health care status of the patient who may be receiving the tissue-engineered composition is emphasized.

Tissue engineering using periosteal cells is a promising approach for bioactive bone repair. Of central importance in tissue engineering is the cell-matrix interaction. In the present study we tested in vitro the influence of alpha-tricalcium phosphate (alpha-TCP) particles on the expression of osteogenic markers in rabbit periosteal cells embedded in specially manufactured fibrin beads. After cell isolation from tibial periosteum of New Zealand White rabbits, and following monolayer culture, cells were embedded in alginate-fibrin beads containing 7.5% alpha-TCP particles and, as a control group, in beads without particles. The alginate was extracted immediately after polymerization. The beads were cultivated for at least 53 days. The DNA content, alkaline phosphatase activity, and osteocalcin level were determined. In monolayer culture the number of cells increased 6.5-fold. DNA content increased in both three-dimensional culture groups but was significantly higher in the beads containing alpha-TCP. Alkaline phosphatase activity increased in both groups without significant differences. Osteocalcin content was significantly higher in the beads containing alpha-TCP than it was in those without alpha-TCP. These observations indicate that matrix engineering using inorganic particles in fibrin culture can influence the osteogenic differentiation of mesenchymal cells. The three-dimensional culture system presented here facilitates the preparation of grafts for bone reconstruction., (Copyright 2001 John Wiley & Sons, Inc. J Biomed Mater Res 59: 690-696, 2002)