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Bone forming ability of recombinant human collagen peptide granules applied with β‐tricalcium phosphate fine particles

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Recombinant human collagen peptide, developed based on human collagen type I, contains an arginyl‐glycyl‐aspartic acid (RGD)‐rich motif to enhance cell behavior and is anticipated as a xeno‐free polymer material for use in tissue engineering. We fabricated granules containing recombinant human collagen peptide (RCP) applied with beta‐tricalcium phosphate fine particles (RCP/β‐TCP) as bone filling scaffold material and assessed the bone forming ability of RCP/β‐TCP. Recombinant peptide was thermal crosslinked and freeze‐dried to prepare RCP. An aqueous dispersion of β‐TCP fine particles was added to RCP to obtain RCP/β‐TCP. Subsequently, RCP/β‐TCP were characterized using scanning electron microscopy (SEM), energy dispersive X‐ray spectrometry (EDX), and cell culture assessments. Furthermore, RCP/β‐TCP were implanted into rat cranial bone defects for radiographic and histological evaluations. In SEM and EDX analyses of RCP/β‐TCP, β‐TCP particles dose‐dependently covered the surface of RCP. Cell culture tests showed that RCP/β‐TCP remarkably promoted proliferation and mRNA expression of various genes, such as integrin β1 and osteogenic markers, of osteoblastic MC3T3‐E1 cells. Histomorphometric assessment at 4 weeks showed that RCP/β‐TCP significantly promoted new skull bone formation compared to RCP (p < 0.05) and control (no application) (p < 0.01). Accordingly, these findings suggest RCP/β‐TCP possess bone forming capability and would be beneficial for bone tissue engineering therapy.
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ORIGINAL RESEARCH REPORT
Bone forming ability of recombinant human collagen peptide
granules applied with β-tricalcium phosphate fine particles
Tomokazu Furihata
1
| Hirofumi Miyaji
1
| Erika Nishida
1
| Akihito Kato
1
|
Saori Miyata
1
| Kanako Shitomi
1
| Kayoko Mayumi
1
| Yukimi Kanemoto
1
|
Tsutomu Sugaya
1
| Tsukasa Akasaka
2
1
Department of Periodontology and
Endodontology, Faculty of Dental Medicine,
Hokkaido University, Sapporo, Japan
2
Department of Biomedical Materials and
Engineering, Faculty of Dental Medicine,
Hokkaido University, Sapporo, Japan
Correspondence
Hirofumi Miyaji, Department of
Periodontology and Endodontology, Faculty of
Dental Medicine, Hokkaido University, N13,
W 7, Kita-ku, Sapporo 060-8586, Japan.
Email: miyaji@den.hokudai.ac.jp
Funding information
AMED Translational Research Program, Grant/
Award Numbers: A105, A154; JPSP KAKENHI,
Grant/Award Numbers: JP16K11822,
JP18K17038
Abstract
Recombinant human collagen peptide, developed based on human collagen type I,
contains an arginyl-glycyl-aspartic acid (RGD)-rich motif to enhance cell behavior and
is anticipated as a xeno-free polymer material for use in tissue engineering. We fabri-
cated granules containing recombinant human collagen peptide (RCP) applied with
beta-tricalcium phosphate fine particles (RCP/β-TCP) as bone filling scaffold material
and assessed the bone forming ability of RCP/β-TCP. Recombinant peptide was ther-
mal crosslinked and freeze-dried to prepare RCP. An aqueous dispersion of β-TCP
fine particles was added to RCP to obtain RCP/β-TCP. Subsequently, RCP/β-TCP
were characterized using scanning electron microscopy (SEM), energy dispersive X-
ray spectrometry (EDX), and cell culture assessments. Furthermore, RCP/β-TCP were
implanted into rat cranial bone defects for radiographic and histological evaluations.
In SEM and EDX analyses of RCP/β-TCP, β-TCP particles dose-dependently covered
the surface of RCP. Cell culture tests showed that RCP/β-TCP remarkably promoted
proliferation and mRNA expression of various genes, such as integrin β1 and osteo-
genic markers, of osteoblastic MC3T3-E1 cells. Histomorphometric assessment at
4 weeks showed that RCP/β-TCP significantly promoted new skull bone formation
compared to RCP (p< 0.05) and control (no application) (p< 0.01). Accordingly, these
findings suggest RCP/β-TCP possess bone forming capability and would be beneficial
for bone tissue engineering therapy.
KEYWORDS
bone filling material, integrin β1, osteogenic differentiation, rat skull, recombinant peptide
based on human collagen type I
1|INTRODUCTION
Improvement of bone tissue engineering therapy is required for the
treatment of bone loss caused by infectious disease, trauma, and can-
cer. Three elements of tissue engineering are proposed (O'Keefe &
Mao, 2011); cells (Diederichs et al., 2010; Fawzy El-Sayed et al., 2015;
Perez et al., 2018), signaling molecules (Kim, Lee, & Kim, 2018;
Peticone et al., 2017; Yin et al., 2018), and natural and artificial
scaffolds (Carrel et al., 2016; Nathanael et al., 2017; Xing et al., 2013)
are essential for bone conductive strategies. Scaffolds play a major
role in stimulating cell proliferation and differentiation and providing
growth and nutrition factors in bone defects (Bose, Roy, &
Bandyopadhyay, 2012). The polymer matrix is widely known as a
good bioabsorbable scaffold material (Hamlet, Vaquette, Shah,
Hutmacher, & Ivanovski, 2017; Sheikh et al., 2016). Especially, colla-
gen type I has great cellular affinity and biodegradability as scaffold
Received: 16 February 2020 Revised: 15 April 2020 Accepted: 18 April 2020
DOI: 10.1002/jbm.b.34632
J Biomed Mater Res. 2020;108B:30333044. wileyonlinelibrary.com/journal/jbmb © 2020 Wiley Periodicals, Inc. 3033
... Combinations with collagen and other types of bioceramics, such as octacalcium phosphate or low-crystalline hydroxyapatite, also showed excellent bone or periodontal tissue forming effects [16e19]. Based on these studies, Furihata et al. prepared RCP granules with b-TCP fine particles (b-TCP/RCP) and showed a significant promotion of osteoblast growth and differentiation, as well as in vivo (in rat) bone formation, compared to normally granulated RCP (without b-TCP) [20]. Therefore, we hypothesized that b-TCP/ RCP represents a promising scaffold material for periodontal tissue engineering. ...
... b-TCP/RCP was fabricated according to the method of Furihata et al. [20]. An RCP solution (7.5%, Cellnest; Fujifilm Wako Pure Chemical Corp., Osaka, Japan) was lyophilized and granulated using a granulator (Quadroco Mill U5; Quadro Engineering, Waterloo, Canada) into particles approximately 1 mm in diameter. ...
... These results suggest that b-TCP/RCP implantation promoted bone healing. Furihata et al. reported that co-culture of b-TCP/RCP and osteoblast-like cells promoted mRNA expression of osteogenic markers (runt-related transcription factor 2, alkaline phosphatase, and bone sialoprotein) to simultaneously stimulate integrin b1 expression [20]. Integrin-mediated cell adhesion to extracellular matrix components, such as type I collagen, is known to activate focal adhesion kinase and its downstream target, bone morphogenetic protein-smad signaling, which is involved in promoting osteoblast differentiation [22]. ...
Article
Objectives: Recombinant human collagen peptide (RCP) is a recombinantly created xeno-free biomaterial enriched in arginine-glycine-aspartic acid sequences with good processability whose use for regenerative medicine applications is under investigation. The biocompatibility and osteogenic ability of RCP granules combined with β-tricalcium phosphate (TCP) submicron particles (β-TCP/RCP) were recently demonstrated. In the present study, β-TCP/RCP was implanted into experimental periodontal tissue defects created in beagles to investigate its regenerative effects. Methods: An RCP solution was lyophilized, granulated, and thermally cross-linked into particles approximately 1 mm in diameter. β-TCP dispersion (1 wt%; 500 μL) was added to 100 mg of RCP granules to form β-TCP/RCP. A three-walled intrabony defect (5 mm × 3 mm × 4 mm) was created on the mesial side of the mandibular first molar and filled with β-TCP/RCP. Results: A micro-computed tomography image analysis performed at 8 weeks postoperative showed a significantly greater amount of new bone after β-TCP/RCP grafting (2.2-fold, P < 0.05) than after no grafting. Histological findings showed that the transplanted β-TCP/RCP induced active bone-like tissue formation including tartaric acid-resistant acid phosphatase- and OCN-positive cells as well as bioabsorbability. Ankylosis did not occur, and periostin-positive periodontal ligament-like tissue formation was observed. Histological measurements performed at 8 weeks postoperative revealed that β-TCP/RCP implantation formed 1.7-fold more bone-like tissue and 2.1-fold more periodontal ligament-like tissue than the control condition and significantly suppressed gingival recession and epithelial downgrowth (P < 0.05). Conclusions: β-TCP/RCP implantation promoted bone-like and periodontal ligament-like tissue formation, suggesting its efficacy as a periodontal tissue regenerative material.
... Based on these studies, Furihata et al. prepared RCP granules with β-TCP fine particles (β-TCP/RCP) and showed a significant promotion of osteoblast growth and differentiation, as well as in vivo (in rat) bone formation, compared to normally granulated RCP (without β-TCP) [30]. Therefore, we hypothesized that β-TCP/RCP represents a promising scaffold material for periodontal tissue engineering. ...
... These results suggest that β-TCP/RCP implantation promoted bone healing. Furihata et al. reported that co-culture of β-TCP/RCP and osteoblast-like cells promoted mRNA expression of the osteogenic markers runt-related transcription factor 2 (Runx2), alkaline phosphatase, and bone sialoprotein, to simultaneously stimulate integrin β1 expression [30]. Integrin-mediated cell adhesion to extracellular matrix components, such as type I collagen, is known to activate focal adhesion kinase and its downstream target, bone morphogenetic protein (BMP)-smad signaling, which is involved in promoting osteoblast . ...
... Accordingly, the replacement of β-TCP/RCP appears to be slower than that of the beta-TCP-blended bovine collagen scaffold previously reported by Ogawa et al. [25]. The slow resorption of β-TCP/RCP was likely caused by the non-connected pore structure (calf-derived collagen sponge has a fully connected pore structure) and a difference in the degree of cross-linking [30]. However, there was almost no accumulation of CD3-positive cells to β-TCP/RCP, suggesting that there is no concern about detrimental inflammatory reactions against periodontal tissue regeneration caused by residual β-TCP/RCP [51]. ...
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Full-text available
Recombinant human collagen peptide (RCP) is a recombinantly created xeno-free biomaterial enriched in RGD (arginine-glycine-aspartic acid) sequences, with good processability that is being investigated for regenerative medicine applications. Recently, the biocompatibility and osteogenic ability of β-TCP/RCP (RCP granules combined with β-tricalcium phosphate (TCP) submicron particles) were demonstrated. In the present study, β-TCP/RCP was implanted into experimental periodontal tissue defects (three-walled bone defect) created in beagle dogs to investigate tissue responses and subsequent regenerative effects. Micro computed tomography image analysis at 8 weeks postoperatively showed that the amount of new bone after β-TCP/RCP graft was significantly greater (2.2 fold, P<0.05) than that of the control (no graft) group. Histological findings showed that the transplanted β-TCP/RCP induced active bone-like tissue formation including TRAP-positive and OCN-positive cells as well as bioabsorbability. Ankylosis did not occur, and periostin-positive periodontal ligament-like tissue formation was observed. Histological measurements revealed that β-TCP/RCP implantation formed 1.7-fold more bone-like tissue and 2.1-fold more periodontal ligament-like tissue than the control, and significantly suppressed gingival recession and epithelial downgrowth (P<0.05). These results suggest that β-TCP/RCP is effective as a periodontal tissue regenerative material.
... Currently, rCols have been investigated for clinical translations in many medical applications, including dermal filler [22], wound dressing [23], drug or cell carrier [24,25], bone void filler [26], and tissue engineering scaffolds [27,28]. After querying the NMPA device database [29], several rColb-MDs have been approved for market entry in China, and the statistic number of products with various expected uses is shown in Fig. 2. ...
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As a class of novel biomaterials manufactured by synthetic biology technologies, recombinant collagens are candidates for a variety of medical applications. In this article, a regulatory scientific perspective on recombinant collagens and their medical devices is presented with a focus on the definition, translation, classification and technical review. Recombinant collagens are categorized as recombinant human collagen, recombinant humanized collagen and recombinant collagen-like protein, as differentiated by specific compositions and structures. Based on their intended uses and associated risks, recombinant collagen-based medical devices are generally classified as Class Ⅱ or Ⅲ in China. The regulatory review of recombinant collagen-based medical devices aims to assess their safety and efficacy demonstrated by scientific evidences generated from preclinical and clinical evaluations. Taken together, opportunities as well as challenges for their future clinical translation of recombinant collagen-based medical devices abound, which highlights the essential role of regulatory science to provide new tools, standards, guidelines and methods to evaluate the safety and efficacy of medical products.
... To date, a series of studies have been conducted on RCP materials at various facilities [27,43,55]. The mechanical properties and biodegradation rate of the bone substitutes influences the quantity and quality of newly formed bone [56]. ...
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Treatment of massive bone defects is one of the most difficult problems to solve in orthopedics. At present, there is no consensus on the best way to resolve these problems. The aim of our study was to evaluate the effect of a three-dimensional bioimplant over massive bone defects, and to analyse if it improves the speed and quality of integration in recipient bone compared to allograft treatment. Fifteen female lambs with massive bone defects, surgically created in their tibias, were randomly divided into three groups of five lambs each: Group I −treated with the bioimplant; Group 2 −treated with the bioimplant plus nucleated cells of autologous bone marrow; Group 3 −treated with a frozen allograft. Radiographs were taken post-treatment at weeks 1, 6, and 12. Animals were euthanized to obtain the studied bone segment for morphological analyses. Treatment: with bioimplants vs. bioimplant plus bone marrow nucleated cells (BMNCs) showed a notorious osteogenic effect, but with greater osteoid synthesis and cellularity in the latter. These results suggest that combined treatment with bioimplants and BMNCs have an additive effect on massive bone defects in lambs. These experimental results could be applied to repair damaged human bone.
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The production of large quantities of functional vascularized bone tissue ex vivo still represent an unmet clinical challenge. Microcarriers offer a potential solution to scalable manufacture of bone tissue due to their high surface area-to-volume ratio and the capacity to be assembled using a modular approach. Microcarriers made of phosphate bioactive glass doped with titanium dioxide have been previously shown to enhance proliferation of osteoblast progenitors and maturation towards functional osteoblasts. Furthemore, doping with cobalt appears to mimic hypoxic conditions that have a key role in promoting angiogenesis. This characteristic could be exploited to meet the clinical requirement of producing vascularized units of bone tissue. In the current study, the human osteosarcoma cell line MG-63 was cultured on phosphate glass microspheres doped with 5% mol titanium dioxide and different concentrations of cobalt oxide (0%, 2% and 5% mol), under static and dynamic conditions (150 and 300 rpm on an orbital shaker). Cell proliferation and the formation of aggregates of cells and microspheres were observed over a period of two weeks in all glass compositions, thus confirming the biocompatibility of the substrate and the suitability of this system for the formation of compact micro-units of tissue. At the concentrations tested, cobalt was not found to be cytotoxic and did not alter cell metabolism. On the other hand, the dynamic environment played a key role, with moderate agitation having a positive effect on cell proliferation while higher agitation resulting in impaired cell growth. Finally, in static culture assays, the capacity of cobalt doping to induce vascular endothelial growth factor (VEGF) upregulation by osteoblastic cells was observed, but was not found to increase linearly with cobalt oxide content. In conclusion, Ti–Co phosphate glasses were found to support osteoblastic cell growth and aggregate formation that is a necessary precursor to tissue formation and the upregaulation of VEGF production can potentially support vascularization.