Strategy to improve endogenous bone regeneration of 3D-printed polycaprolactone/hydroxyapatite composite scaffold: Collagen designs with bone morphogenetic protein 2 and fibroblast growth factor 2

Strategy to improve endogenous bone regeneration of 3D-printed polycaprolactone/hydroxyapatite composite scaffold: Collagen designs with bone morphogenetic protein 2 and fibroblast growth factor 2 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Strategy to improve endogenous bone regeneration of 3D-printed polycaprolactone/hydroxyapatite composite scaffold: Collagen designs with bone morphogenetic protein 2 and fibroblast growth factor 2 Yong Sang Cho, Min-Soo Ghim, Myoung Wha Hong, Young Yul Kim, Young-Sam Cho This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1903800/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In bone tissue engineering, the endogenous regeneration of bone defects still represents a clinical challenge despite the development of intervention therapy to achieve bone regeneration via autologous grafts, allogeneic grafts, bone morphogenetic protein (BMP)-2, etc. To overcome the limitation of endogenous bone regeneration, we assumed that the 3D-printed collagen pattern with BMP-2 and fibroblast growth factor (FGF)-2 in the 3D-printed polycaprolactone/nano-hydroxyapatite scaffold could guide the endogenous regeneration of bone defects. Therefore, to test our hypothesis, polycaprolactone/nano-hydroxyapatite/collagen scaffolds with dual growth factors (BMP-2 and FGF-2) and various hydrogel patterns (positive, edge, and radial patterns) were fabricated at the same ratio and concentration of dual growth factors. Consequently, we revealed that the in vitro released concentrations of BMP-2 and FGF-2 were not affected by collagen patterns in the PCL/nano-hydroxyapatite scaffold. Furthermore, endogenous bone regeneration and angiogenesis in the polycaprolactone/nano-hydroxyapatite/collagen scaffold with a radial pattern were promoted compared with those in the polycaprolactone/nano-hydroxyapatite/collagen scaffolds with positive and edge patterns. Therefore, we demonstrated that a collagen design loaded with dual growth factors in the 3D-printed scaffold could affect endogenous bone regeneration and angiogenesis. Bone tissue engineering 3D printing Polymer-matrix composites Collagen Hybrid scaffold. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Large bone defects with critical defect sizes may occur frequently due to traffic accidents, trauma, tumors (osteosarcoma), or congenital diseases in humans. In terms of bone regeneration, the endogenous regeneration of bone defects still represents a clinical challenge despite the development of intervention therapy to achieve bone regeneration via autologous grafts, allogeneic grafts, BMP-2, etc. [ 1 – 4 ]. The current intervention therapy using bone grafts may lead to drawbacks reported in the literature, such as the risk of disease transmission and immunogenic response, scarcity of bone donors, or discrepancy due to the limited shape of powder or block types [ 5 – 7 ]. In bone tissue engineering, a 3D synthetic scaffold with biocompatible and biodegradable properties, which has been developed as an alternative to conventional bone grafts, is regarded to play a key role along with cells and growth factor proteins associated with the improvement of bone cell differentiation and maturation [ 8 , 9 ]. In the fabrication of a 3D synthetic scaffold, 3D printing has been highlighted because it can fabricate a customized 3D synthetic scaffold with a favorable design for target tissues and cells, including pore size, porosity, and geometrical characteristics, using computer-aided design/computer-aided manufacturing techniques (CAD/CAM) [ 10 , 11 ]. Many studies have focused on enhancing bone defect recovery using osteoconductive 3D synthetic scaffolds and osteoinductive growth factors. Osteoconduction refers to bone growth on the surface of a graft. Osteoinduction involves the recruitment of immature cells and stimulation of these cells to develop into preosteoblast cells [ 12 ]. In 3D-printing technique, to improve the osteoconduction ability and mechanical properties of 3D synthetic scaffolds, composite materials consisting of synthetic polymers (PCL, PLA, PVA, etc.) and bioceramics (HA, β-TCP, bioactive glass, etc.) have been reported in literature for bone regeneration [ 13 – 17 ]. To enhance the osteoinduction ability of 3D synthetic scaffolds, hybrid (synthetic polymer/ hydrogel) or hydrogel scaffolds loaded on bone morphogenetic proteins (BMPs) have been proposed [ 18 – 20 ]. From the viewpoint of the endogenous regeneration of large bone defects, the formation of vascular networks by angiogenic growth factors has attracted attention as a strategy for large bone regeneration. The formation of a vascular network within the bone constructs could provide essential nutrients and oxygen via diffusion to the bone tissue or cells [ 21 , 22 ]. To achieve endogenous regeneration of large bone defects, a 3D synthetic scaffold with dual growth factors consisting of BMPs and angiogenic (VEGF, fibroblast growth factor (FGF)-2, PDGF, etc.) growth factors has been reported in the literature [ 23 , 24 ]. According to previous studies [ 25 – 27 ], the ratio, concentration, and type of BMPs and angiogenic growth factors influence the bone-regenerating ability of 3D synthetic scaffolds. We assumed that the 3D-printed collagen pattern with BMP-2 and FGF-2 growth factors in the 3D-printed 3D synthetic scaffold could be guided in the endogenous regeneration of bone defects. Therefore, in this study, to demonstrate our hypothesis, the PCL/nHA composite material with relatively enhanced osteoconduction and mechanical properties was used for the fabrication of 3D-printed 3D synthetic scaffolds. The kagome structure of the PCL/nHA scaffold was selected as a delivery system for growth factors because a scaffold with a kagome structure can be adjusted to have various collagen designs via an 3D-printing system. Moreover, the kagome structure can enhance the mechanical properties of the scaffold, including the compressive modulus, tensile modulus, and bending modulus, compared with those of the structure of a conventional scaffold fabricated using an 3D-printing system [ 8 , 28 , 29 ]. PCL/nHA/collagen scaffolds with dual growth factors (BMP-2 and FGF-2) and various hydrogel patterns (positive, edge, and radial patterns) were fabricated at the same ratio and concentration of dual growth factors. The possibility of endogenous regeneration of bone defects was investigated using the aforementioned scaffold. 2. Materials And Methods 2.1 Preparation and fabrication of the PCL/nHA/collagen scaffold with dual growth factors (FGF-2 and BMP-2) Commercial PCL (M w = 43,000–50,000 Da, Polysciences, Warrington, PA, USA) and nHA (particle size: < 200 nm, Sigma-Aldrich, St. Louis, MO, USA) were purchased to prepare the composite material. The PCL/nHA composite material (PCL/nHA) was prepared as described in our previous studies [ 8 , 10 ]. Briefly, PCL pellets were dissolved in 5% (w/v) dichloromethane (DCM; Daejung Chemicals and Materials, Siheung, Republic of Korea) using a magnetic stirrer for 1 h at 500 rpm. nHA powder (10 wt%) was added to a 5 w/v% PCL/DCM solution and dried in a vacuum oven at 80°C for 72 h. The dried PCL/nHA composite material was melted at 88°C for 1 h in a dispenser with the inner diameter of the dispenser ceramic nozzle being 100-µm. Simultaneously, to inject collagen with dual growth factors in the fabricating PCL/nHA scaffold with a kagome structure, 500 µL collagen type I (Concentration 3 w/v%) (COLTRIX®, Ubiosis, Seongnam, Republic of Korea), 50 µL BMP-2 solution of 2 µg/µL concentration (Recombinant Human/Murine/Rat BMP-2, PeproTECH, Rocky Hill, NJ, USA) solubilized in sterile water, and 50 µL FGF-2 solution of 1 µg/µL concentration (Recombinant Human FGF-basic, PeproTECH, Rocky Hill, NJ, USA) in 5 mM Tris buffer were carefully mixed by pipetting. The prepared collagen was injected into a 100 µL syringe (Gastight-type model 1705, Hamilton, Reno, NV, USA). Subsequently, the syringe was equipped with an 3D-printing system (lab-made extrusion type) (Fig. 1 ). As an early step in the fabrication process of the PCL/nHA/collagen scaffold, a 3D scaffold with a kagome structure was drawn via composite-material extrusion by rotating a single screw at 45 rpm under an air pressure of 250 kPa. Second, after the composite-material extrusion process, collagen droplets (0.5 µL or 1 µL) were injected into the pores of the 3D scaffold with a kagome structure according to design patterns, including positive, edge, and radial patterns, via the collagen-extruding system (Fig. 1 (a)). For the loading amount of BMP-2 and FGF-2 in the fabricated PCL/nHA/collagen scaffold, the loading amounts of each scaffold with various collagen patterns were calculated as 2-µg BMP-2 and 1-µg FGF-2. The parameters of the fabricated PCL/nHA/collagen scaffolds are presented in Table 1 . Furthermore, the total volume of the injected collagen was adjusted to 13 µL for each scaffold with various collagen patterns. For gelation of the injected collagen in the fabricated scaffold, the PCL/nHA/collagen scaffold was placed in an incubator for 30 min at 37°C with 95% air and 5% CO 2 . In addition, to compare bone formation of the scaffold with various collagen patterns, PCL/nHA/collagen scaffolds with positive, edge, and radial patterns were fabricated (Fig. 1 (b)). In terms of the in vitro and in vivo experiments, the cubic-shape and plug-shape scaffolds with a dimension of 5 mm × 5 mm × 3.6 mm and D 1 :8.9 mm/D 2 :7.9 mm/H:1.3 mm were fabricated, respectively (Fig. 1 (a)). Table 1 Design parameters of 3D-printed PCL/nHA/collagen scaffold Parameter PCL/nHA/collagen (positive pattern) PCL/nHA/collagen (edge pattern) PCL/nHA/collagen (radial pattern) Porosity 50.6 ± 0.5% 49.5 ± 0.9% 49.9 ± 0.6% Pore size 510 ± 22 µm 509 ± 17 µm 513 ± 29 µm Calculating total volume of collagen 13 µl 13 µl 13 µl Calculating amount of BMP-2 loaded 2 µg 2 µg 2 µg Calculating amount of FGF-2 loaded 1 µg 1 µg 1 µg 2.2 Design of the PCL/nHA/collagen scaffold with kagome structure To fabricate a PCL/nHA/collagen scaffold with a kagome structure using an 3D-printing system, the kagome structure was designed as reported in literature [ 29 ]. The designed scaffold had the following structural characteristics: approximate porosity of 50% and pore size of 500 µm. The 3D-printing pathway based on the g-code was generated using slicing software (Slic3r, version 1.2.9) via the standard tessellation language file of the designed scaffold. 2.3 Scaffold characterization The porosities of the PCL/nHA (10 wt%) scaffolds without collagen were calculated using Eq. ( 1 ) as follows (a total of 10 scaffolds were used): $$\text{Porosity }\left(\%\right) = \frac{{v}_{1}-\left(\frac{\text{0.9}\times m}{{\rho }_{\text{PCL}}}+\frac{0.1\times m}{{\rho }_{\text{HA}}}\right)}{{v}_{1}}\times 100$$ 1 where \({v}_{1}\) is the calculated apparent volume of the fabricated PCL/nHA scaffold via the scaffold’s outer dimension, ρ PCL and ρ HA are the densities of PCL and nHA, respectively, and m is the weight of the fabricated scaffold. To compare the pore size of the fabricated PCL/nHA scaffold without collagen, the vertical and horizontal distances of the apparent pores on the surface of the scaffold were averaged using an optical microscope (MI-9100 ZOOM, Magic i, Seoul, Republic of Korea). Repetitive experiments were performed for each type via 10 scaffolds. Chemical analysis of the pure PCL, nHA, and PCL/nHA scaffolds with various nHA weight ratios (3, 5, and 10 wt%) was performed using Fourier transform infrared spectroscopy (FT-IR; 6300FV, JASCO, Tokyo, Japan). Repetitive experiments were performed for each sample type using two samples. Moreover, the mass of the fabricated scaffold’s residual material from 30 to 600°C was investigated using TGA (SDT Q600, TA instruments, New Castle, DE, USA). Three scaffolds were used for each type. To determine the tensile modulus of PCL/nHA bulk with various nHA weight ratios (3, 5, and 10 wt %) and PCL/nHA scaffolds with various nHA weight ratios (3, 5, and 10 wt%), tensile tests were conducted (UTM; MTS, Eden Prairie, MN, USA) at a constant strain rate of 1 mm/ min with a 5 kN loading cell. For the tensile test, dumbbell-shaped composite bulk and PCL/nHA scaffolds were fabricated according to the ISO standard (KS M ISO 527-4:2002). The tensile moduli of the PCL/nHA bulk and PCL/nHA scaffold were determined in the linear region within 1% strain of the stress-strain (S-S) curve. Repetitive experiments were performed for each sample type using eight samples. 2.4 Release profile of BMP-2 and FGF-2 The release of BMP-2 and FGF-2 from the prepared PCL/nHA/collagen scaffold in the culture medium was analyzed by ELISA (R&D Systems, Minneapolis, MN, USA). The prepared PCL/nHA/collagen scaffold was placed in 2 ml Dulbecco’s modified Eagle’s medium (DMEM; Gibco, New York, NY, USA), and supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 µg/mL streptomycin, and 100 U/ mL penicillin (Gibco, Carlsbad, CA, USA). The cells were maintained in an incubator at 37°C and 5% CO 2 . The culture medium was collected for 3 weeks. The concentrations of released BMP-2 and FGF-2 growth factors were calculated according to the ELISA kit protocols. 2.5 Human osteoblast-like cell culture and proliferation Cultured human osteogenic sarcoma cells after a few passages (Saos-2 cells; Korea Cell Line Bank, Seoul, Republic of Korea) were cultured in DMEM (Gibco, New York, NY, USA), and supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 100 µg/mL streptomycin, and 100 U/mL penicillin (Gibco, Carlsbad, CA, USA) in a humidified incubator at 37°C with 5% CO 2 . The prepared PCL/nHA scaffolds were sterilized by immersion in 70% EtOH overnight under UV light. The cells in suspension (1×10 5 cells/scaffold) were seeded onto the surface of the prepared PCL/nHA scaffolds. Cultures of cell-seeded scaffolds were maintained at 37°C with 5% CO 2 . To evaluate cell adhesion and proliferation in the scaffold, qualitative cell numbers were determined using a cell counting kit (CCK-8; Dojindo, Kumamoto, Japan) at an absorbance of 450 nm using a multimode plate reader after culturing for 1, 7, and 14 days. The viability of human osteoblast-like cells on the scaffolds was investigated using a live/dead kit (Molecular Probes, Eugene, OR, USA). 2.6 Scaffold implantation without cells in rat’s calvarial defect for in vivo study The in vivo study was performed according to all procedures and principles approved by the Institutional Review Board of St. Mary’s Hospital of Catholic University (CMCDJ-AP-2019-005). To anesthetize eight-week-old SD rats ( n = 5), intraperitoneal injections of ketamine hydrochloride (Yuhan, Seoul, Republic of Korea) and Rompun® (Bayer, Leverkusen, Germany) were administered. After anesthetized SD rats were placed on a heated pad, the surgical site of SD rats was shaved and sterilized with povidone. The rats were placed on an operating table with a heated pad and covered with a sterile drape. After a longitudinal midline incision was made, the periosteum and skin were carefully detached. To create a circular defect with a diameter of 8-mm in the calvarial bone, an 8-mm trephine bur was utilized. Each PCL/nHA/collagen scaffold with positive, edge, and radial patterns was implanted into a calvarial defect. To finish the scaffold implantation, the cleaved skin was sutured with 4 − 0 black silk (Ailee, Busan, Republic of Korea). To investigate bone formation by scaffold implantation in the calvarial defect, SD rats were sacrificed in the eighth week after implantation. Calvarial samples were harvested from sacrificed SD rats. The cells were then fixed with 10% formalin. All the surgical procedures were performed under aseptic conditions. 2.7 Quantitative analysis of the bone formation by PCL/nHA/collagen scaffold via µCT To quantitatively analyze bone formation in the scaffolds, the harvested calvarial samples were scanned using a micro-CT (µCT) scanner (Skyscan 1172, Bruker, Billerica, MA, Belgium). The harvested calvarial samples were scanned (resolution: 13 µm and aluminum filter: 500 µm), and micro-radiographic images were obtained at 60 kV and 167 µA. To reconstruct the harvested calvarial samples, the 6-bit scanning files obtained were converted into DICOM files (DicomCT version 2.5, Bruker, Billerica, MA, USA). The harvested calvarial samples were graphicalized using prepared DICOM files and reconstruction software (Mimics version 21.0, Materialise, Leuven, Belgium). For this 3D remodeling, the threshold values (lower and upper) for natural bone were presumed to be 95 and 250 Hounsfield units. The total bone volume within the cylindrical region of interest (ROI; \(8\times 1\text{ mm}\) or \(4\times 1\text{ mm}\) ) was measured by assigning an assumed threshold value for the total bone content. Five harvested calvarial samples were analyzed in each group. All scanning and 3D remodeling parameters were identical in all the groups. Moreover, all µCT images were selected that were similar to the average values of the measured bone volume. 2.8 Preparation for histological and immunohistochemistry (IHC) staining Specimens for histological and immunohistochemical analyses were prepared after scanning harvested calvarial samples. Fixation and decalcification of harvested calvarial samples were performed by immersion in 10% formalin and 10% nitric acid solution. After fixation and decalcification, calvarial samples were embedded in paraffin. The calvarial samples were embedded in paraffin. Thereafter, the slides of calvarial samples were stained with hematoxylin and eosin (H&E) and Masson’s trichrome stains after the paraffin blocks were cut to 4-µm thicknesses. Masson’s trichrome staining was performed according to the manual of the Masson’s trichrome stain kit (Polysciences, Warrington, PA, USA). For immunohistochemical staining, paraffin tissue slides were dissolved in xylene to remove the paraffin. The deparaffinized tissue slides were rehydrated using serial concentrations of ethanol. After washing, rehydrated tissue slides were retrieved using citrate buffer. Thereafter, the tissue slides were blocked for 30 min and incubated with a rat anti-CD31 antibody (Novus biologicals, Littleton, CO, USA) overnight at 4°C. The tissue slides were incubated with secondary antibodies for 1 h and observed using an ImmPACT ® NovaRED ® substrate peroxidase (SK-4805; Vector Laboratories, Burlingame, CA, USA). 2.9 Statistical analysis Statistical analysis was performed via Student’s t-test (Microsoft 2019, Redmond, WA, USA) using the measured data. Student’s t-test was performed considering a value of P < 0.05. The obtained data are presented as the mean ± standard deviation. 3. Results 3.1 Evaluation of in vitro characteristics for the fabricated PCL/nHA scaffolds without collagen at a specific ratio of nHA To compare the characteristics of the fabricated PCL/nHA scaffolds with a kagome structure, the HA weight ratio, tensile modulus, and in vitro cell response were investigated. The nHA amounts in the fabricated PCL/nHA scaffolds were determined via FT-IR and TGA before evaluating the characteristics of the fabricated PCL/nHA scaffolds. We revealed that the detection level of the P-O group, which is a representative chemical component of nHA, increased with the nHA weight ratio of the fabricated scaffolds (Fig. 2 (a) and (b)). Moreover, the PCL and PCL/nHA (3, 5, and 10 wt%) scaffolds’ residuals at 600°C were measured as 0.4 ± 0.1%, 3.7 ± 0.6%, 5.4 ± 0.7%, and 10.1 ± 0.2% (Fig. 2 (c)). We verified that the actual nHA amount in the fabricated scaffolds was similar to the designed HA weight ratio of the PCL/nHA scaffold. The tensile moduli of PCL and PCL/nHA (3, 5, and 10 wt%) bulks were 327.7 ± 2.0 MPa, 334.5 ± 12.1 MPa, 340.0 ± 7.7 MPa, and 398.7 ± 15.6 MPa, respectively. The tensile moduli of PCL and PCL/nHA (3, 5, and 10 wt%) scaffolds were found to be 86.8 ± 4.5 MPa, 97.3 ± 3.2 MPa, 106.6 ± 3.5 MPa, and 121.7 ± 9.8 MPa, respectively. In terms of in vitro osteoblast-like cell response, the proliferation and viability of cells cultured on fabricated PCL/nHA scaffolds for 2 weeks were investigated at the nHA’s specific ratio (Fig. 3 ). The cell proliferation of the PCL/nHA scaffold with 10 wt% nHA was higher than that of other scaffolds, although there was no difference in the cell adhesion of the fabricated PCL/nHA scaffolds with increasing nHA weight ratio (Fig. 3 (a) and (b)). Moreover, the numbers of dead cells on the surface of the PCL/nHA scaffold with 10 wt% were less than that on the other scaffolds. 3.2 Assessment of release profiles of loaded dual growth factors and bone formation of PCL/nHA/collagen scaffolds with various collagen patterns The release trends of dual growth factors (BMP-2 and FGF-2) loaded on the PCL/nHA/collagen scaffolds were investigated according to the proposed collagen patterns (Fig. 4 ). In terms of BMP-2 release (Fig. 4 (a)), the release trend of BMP-2 from the PCL/nHA/collagen scaffold with a positive pattern was similar to the radial pattern over 21 days. Although BMP-2 release from the PCL/nHA/collagen scaffold with edge pattern was slightly different compared with the positive and radial patterns before 7 days, the released concentration of the PCL/nHA/collagen scaffold with edge pattern was similar to that of other scaffolds after 14 days. Furthermore, the release trends of FGF-2 from all groups were virtually the same over 21 days (Fig. 4 (b)). Consequently, the total concentrations of dual growth factors released from the prepared scaffolds with various collagen patterns were similar. For quantitative analysis of bone formation via micro-CT (Fig. 5 ), the average bone volumes in accordance with 8-mm ROI in the calvarial defect model without scaffold and the PCL/nHA/collagen scaffold with various patterns (positive, edge, and radial patterns) were measured as 4.6 ± 3.3 mm 3 , 15.0 ± 3.4 mm 3 , 14.0 ± 6.2 mm 3 , and 27.4 ± 3.9 mm 3 , respectively. The bone-regenerating levels of all the groups for 4-mm ROI were investigated as 0.0 ± 0.0 mm 3 , 0.6 ± 0.5 mm 3 , 0.7 ± 0.9 mm 3 , and 5.0 ± 0.4 mm 3 , respectively. The bone-regenerating level of all scaffolds was superior to that of the calvarial defect model without scaffold at 8 weeks after implantation. Furthermore, we revealed that the bone volumes were different between scaffold groups based on collagen patterns with dual growth factors at the same concentration and total volume loaded with collagen. 3.3 Histological evaluation of bone formation and angiogenesis in the fabricated scaffolds Histological and IHC analyses for newly formed bone and blood vessels were performed using H&E, Masson’s trichrome, and IHC staining for CD31 antibody at 8 weeks after implantation (Figs. 6 – 9 ). For H&E and Masson’s trichrome staining, the tissues in the calvarial defect model without the scaffold were relatively loose (Figs. 6 and 7 ). In contrast, matured bone tissues were observed in all scaffold groups compared to that seen in the calvarial defect model without scaffold (Figs. 6 , 7 , and 9 ). Furthermore, the matured bone tissues in the PCL/nHA/collagen scaffolds with a radial pattern were higher than those in the other scaffolds. For immunohistochemical staining, formation of numerous blood vessels in scaffold groups was identified compared with that seen in the calvarial defect model without scaffold (Figs. 8 and 9 ). The formation of blood vessels in the mature bone tissue of the PCL/nHA/collagen scaffold with a radial pattern was greater than that of the other scaffolds (Fig. 9 ). The action level of the CD 31 antibody between the scaffold groups was similar. 4. Discussion To investigate the influence of various collagen patterns loaded with dual growth factors (BMP-2 and FGF-2) in the implanted PCL/nHA/collagen scaffold on endogenous bone formation and angiogenesis, the pore size, porosity, concentration of dual growth factors, and total volume of loaded collagen were fixed for each type, as shown in Table 1 . In terms of pore size and porosity, an interconnected pore size of 300–500 µm and 50% porosity are recommended to promote bone formation and vascularization in the 3D scaffold reported in the literature [ 30 – 32 ]. Therefore, the pore size and porosity of the scaffold with a kagome structure were fixed at 500 µm and 50%, respectively, to exclude the effect of the geometric parameters of the scaffold on the bone-regenerating ability. For the weight ratio of the PCL/nHA composite material, 10 wt% of nHA was considered as the maximum weight ratio because the composite material with over 10 wt% nHA could have disadvantages, including non-uniform extrusion and nozzle clogging. Regarding the concentration of BMP-2 and FGF-2, BMP-2 has been known to have an important role in osteogenesis; for example, it is involved in the differentiation of mesenchymal stem cells to osteoblasts or chondrocytes for bone recovery [ 23 , 33 ]. FGF-2 can promote angiogenesis. Generally, angiogenesis is considered an essential process for recovery from damaged bone. Moreover, newly formed blood vessels can provide nutrients, growth factors, and stem cells to the damaged bone site [ 34 , 35 ]. Therefore, we selected BMP-2 and FGF-2 as the growth factors for bone recovery. From the viewpoint of the amount and concentration of dual growth factors, Kim et. al. reported that bone formation induced by BMP-2 did not increase when the injection amount exceeded 1-µg in a mouse calvarial defect model [ 19 ]. Moreover, Liu et. al. reported that the bone-regenerating activity of a 2:1 weight ratio (BMP-2:FGF-2) was superior to other weight ratios for BMP-2 and FGF-2 (1:1, 2:1, 4:1, and 8:1) [ 34 ]. Tabata et. al. verified the synergistic effect of BMP-2/FGF-2 (2:1 weight ratio) and BMP-2/VEGF (3:1 weight ratio), which are known to be suitable weight ratios of dual growth factors for bone regeneration [ 35 ]. The synergistic effect of BMP-2/FGF-2 (2:1 weight ratio) was better than that of BMP-2/VEGF (3:1 weight ratio) in bone regeneration and vascular formation. In this study, the BMP-2 amount loaded onto the scaffold with a kagome structure was fixed at 2 µg. Furthermore, to maximize the synergistic effect of BMP-2 and FGF-2, the weight ratio was fixed at 2:1 (BMP-2:FGF-2). For the hydrogel material, the collagen type I was selected as a carrier system of the dual growth factors because bone tissues are primarily composed of 70% mineral (primarily hydroxyapatite), 22% protein (primarily collagen type I), and 8% other components [ 36 , 37 ]. For the in vitro experiment, a change in the mechanical properties of a composite bulk/3D-printed scaffold and the human osteoblast-like cell response of the 3D scaffold were investigated according to the increasing weight ratio of nHA in the PCL/nHA composite material before the in vivo experiment using a rat calvarial defect model. To verify the actual weight ratio of the nHA powder in the prepared PCL/nHA composite material, changes in the chemical components and amount of residual material at 600°C were measured because PCL was incinerated at approximately 450°C (Figs. 2 (a-c)). The detection level of the P-O group increased with the nHA weight ratio in the PCL/nHA scaffolds. In contrast, the P-O group was not detected in the pure PCL and PCL scaffolds (Figs. 2 (a) and (b)). Moreover, the actual amount of nHA powder was similar to the designed weight ratio of nHA powder in the PCL/nHA composite material (Fig. 2 (c)). With respect to the tensile modulus of bulk and 3D-printed scaffolds, the tensile modulus of the PCL bulk was similar to that of the PCL/nHA bulk (3 and 5 wt%). However, the tensile modulus of the PCL/nHA bulk (10 wt%) was higher than that of the other bulk materials (Fig. 2 (d)). The tensile modulus of the PCL/nHA scaffold was enhanced by increasing the nHA weight ratio. Specifically, the tensile modulus of the PCL/nHA scaffold (10 wt. %) was superior to that of the other scaffolds. Moreover, with respect to the response of human osteoblast-like cells, the cell-adhesion abilities of the PCL and PCL/nHA scaffolds with various weight ratios of nHA did not differ significantly (Figs. 3 ). This phenomenon could explain why the weight ratio of nHA in the PCL/nHA composite material did not influence cell adhesion. However, the cell growth abilities of PCL/nHA scaffolds with various weight ratios were higher than those of PCL scaffolds at 7 and 14 days after cell culture on the fabricated scaffold because of the osteoconductive property of nHA. Furthermore, the cell growth level of the PCL/nHA scaffold with 10 wt% nHA was superior to that of other scaffolds after 7 days (Figs. 3 (a) and (b)). The number of dead cells on the PCL/nHA scaffold with 10 wt% nHA was less than that on the other scaffolds (Fig. 3 (c)). Consequently, to investigate endogenous bone regeneration and angiogenesis by various collagen design patterns with dual growth factors, the PCL/nHA scaffold with 10 wt% nHA was selected because it has enhanced mechanical properties and higher cell response compared to that of other scaffolds. Additionally, to compare the release trends of dual growth factors from the PCL/nHA/collagen scaffolds with various patterns, cumulative release was measured over 21 days (Fig. 4 ). For the release profile of BMP-2, although the concentration of BMP-2 released from PCL/nHA/collagen with an edge pattern was different compared to the positive and radial patterns, the released concentrations of BMP-2 from all scaffolds were saturated after 7 days (Fig. 4 (a)). Furthermore, the overall trends of released FGF-2 from all scaffolds were similar for 21 days (Fig. 4 (b)). This result indicated that the proposed collagen patterns did not affect the release profile of the dual growth factors. For the in vivo experiment using the rat calvarial defect model, to assess endogenous bone regeneration and angiogenesis, the analysis area was divided from the edge and central areas (Figs. 5 – 8 ). From the micro-CT results, the bone volumes of all scaffolds in the 4-mm and 8-mm ROI were superior to those of the calvarial defect model (Fig. 5 (e)). The bone formation level of the PCL/nHA/collagen scaffold with a radial pattern in the 8-mm ROI was higher than those of other scaffolds. Specifically, the bone volume of the PCL/nHA/collagen scaffold with a radial pattern in the 4-mm ROI was significantly higher than that of the other scaffolds. This phenomenon could explain why the existence or nonexistence of space for endogenous bone regeneration, along with dual growth factors, affected bone regeneration. In other words, a collagen design loaded with dual growth factors in the scaffold could affect endogenous bone regeneration. For the H&E- and Masson’s trichrome–stained sections, matured bone tissue in all scaffolds was strongly expressed compared to that in the calvarial defect model (Figs. 6 and 7 ). In the edge area, many bone tissues were newly formed in all scaffolds. However, in the central area, mature bone tissue in the PCL/nHA/collagen scaffold with a radial pattern was more abundant than that in the other scaffolds (Figs. 6 and 7 ). This result was in accordance with the micro-CT results. For IHC and staining, the expression levels of endothelial cells in all scaffolds were higher than those in the calvarial defect model (Figs. 8 and 9 ). The expression levels of endothelial cells did not differ between the scaffold groups. However, many blood vessels in the newly formed tissues of the PCL/nHA/collagen scaffold with a radial pattern were already observed compared to that seen with other scaffolds (Fig. 9 (c)). Consequently, we demonstrated that a collagen design loaded with dual growth factors in the 3D-printed scaffold could affect endogenous bone regeneration and angiogenesis. Therefore, the provision of space for bone regeneration must be considered seriously when collagen design contains a dual growth factor in the 3D-printed scaffold. In our opinion, the clinical problem of endogenous regeneration of bone defects can be overcome by the 3D-printed scaffold in consideration of the abovementioned design parameters. 5. Conclusion In this study, to evaluate the influence of collagen patterns with dual growth factors in the PCL/HA scaffold on endogenous bone regeneration and angiogenesis, PCL/nHA/collagen scaffolds with kagome structure and various collagen patterns (positive, edge, and radial patterns) were fabricated using an 3D-printing system (material extrusion type). In terms of nHA weight ratio, the mechanical properties and human osteoblast-like cell response of the PCL/nHA scaffold with 10 wt% nHA were superior to those of the other scaffolds. We found that the in vitro released concentrations of BMP-2 and FGF-2 were not affected by collagen patterns in the PCL/nHA scaffold with 10 wt% nHA. Furthermore, endogenous bone regeneration and angiogenesis in the PCL/nHA/collagen scaffold with a radial pattern were promoted compared with those in the PCL/nHA/collagen scaffolds with positive and edge patterns. Therefore, we demonstrated that a collagen design loaded with dual growth factors in the 3D-printed scaffold could affect endogenous bone regeneration and angiogenesis. In other words, a design that considers the supply of space for bone regeneration is important when collagen design incorporates dual growth factors in the 3D-printed scaffold for effective endogenous regeneration in bone tissue engineering. Declarations Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2021R1A2C2009665). Conflict of Interest statement The authors declare no conflict of interest. Author’s contribution Conceptualization, Yong Sang Cho and Young-Sam Cho, methodology, Yong Sang Cho, Myoung Wha Hong, and Young Yul Kim, software, Yong Sang Cho and Min-Soo Ghim, writing-original draft preparation, Yong Sang Cho, Min-Soo Ghim, and Myoung Wha Hong, writing-review and editing, Yong Sang Cho and Young-Sam Cho, supervision, Young Yul Kim and Young-Sam Cho. References Perier-Metz C, Duda GN, Checa S (2020) Mechano-Biological Computer Model of Scaffold-Supported Bone Regeneration: Effect of Bone Graft and Scaffold Structure on Large Bone Defect Tissue Patterning. 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Macromol Biosci 10:12–27 Koria P (2012) Delivery of growth factors for tissue regeneration and wound healing. BioDrugs 26:163–175 Hajimiri M, Shahverdi S, Kamalinia G, Dinarvand R (2015) Growth factor conjugation: Strategies and applications. J Biomed Mater Res Part A 103:819–838 Park JY, Shim JH, Choi SA, Jang J, Kim M, Lee SH et al (2015) 3D printing technology to control BMP-2 and VEGF delivery spatially and temporally to promote large-volume bone regeneration. J Mater Chem B 3:5415–5425 Cai WX, Zheng LW, Li CL, Ma L, Ehrbar M, Weber FE et al (2014) Effect of different rhBMP-2 and TG-VEGF ratios on the formation of heterotopic bone and neovessels. Biomed Res Int 2014:571510 Charles LF, Woodman JL, Ueno D, Gronowicz G, Hurley MM, Kuhn LT (2015) Effects of low dose FGF-2 and BMP-2 on healing of calvarial defects in old mice. Exp Gerontol 64:62–69 Lee SH, Cho YS, Hong MW, Lee BK, Park Y, Park SH et al (2017) Mechanical properties and cell-culture characteristics of a polycaprolactone kagome-structure scaffold fabricated by a precision extruding deposition system. Biomed Mater 12:055003 Cho YS, Quan M, Kang NU, Jeong HJ, Hong MW, Kim YY et al (2020) Strategy for enhancing mechanical properties and bone regeneration of 3D polycaprolactone kagome scaffold: Nano hydroxyapatite composite and its exposure. Eur Polym J 134:109814 Limmahakhun S, Oloyede A, Sitthiseripratip K, Xiao Y, Yan C (2017) 3D-printed cellular structures for bone biomimetic implants. Addit Manuf 15:93–101 Wang P, Li X, Jiang Y, Nai MLS, Ding J, Wei J (2020) Electron beam melted heterogeneously porous microlattices for metallic bone applications: Design and investigations of boundary and edge effects. Addit Manuf 36:101566 Kim D, Lee JU, Kim GH (2020) Biomimetic gelatin/HA biocomposites with effective elastic properties and 3D-structural flexibility using a 3D-printing process. Addit Manuf 36:101616 Chen R, Wang J, Liu C (2016) Biomaterials Act as Enhancers of Growth Factors in Bone Regeneration. Adv Funct Mater 26:8810–8823 Wang L, Huang Y, Pan K, Jiang X, Liu C (2010) Osteogenic responses to different concentrations/ratios of BMP-2 and bFGF in bone formation. Ann Biomed Eng 38:77–87 Kuttappan S, Mathew D, Jo JI, Tanaka R, Menon D, Ishimoto T et al (2018) Dual release of growth factor from nanocomposite fibrous scaffold promotes vascularisation and bone regeneration in rat critical sized calvarial defect. Acta Biomater 78:36–47 Ferreira AM, Gentile P, Chiono V, Ciardelli G (2012) Collagen for bone tissue regeneration. Acta Biomater 8:3191–3200 Agrawal V, Sinha M (2017) A review on carrier systems for bone morphogenetic protein-2. J Biomed Mater Res Part B Appl Biomater 105:904–925 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1903800","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":127805508,"identity":"de4db821-18b5-42b3-a1a0-f70abbd46f48","order_by":0,"name":"Yong Sang Cho","email":"","orcid":"","institution":"TE BioS Co., Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yong","middleName":"Sang","lastName":"Cho","suffix":""},{"id":127805509,"identity":"6aa0ec13-e16e-4745-9868-3587207756ee","order_by":1,"name":"Min-Soo Ghim","email":"","orcid":"","institution":"Wonkwang University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Min-Soo","middleName":"","lastName":"Ghim","suffix":""},{"id":127805510,"identity":"2ba9f30c-abeb-4033-8d2a-34ef8cb1e6cc","order_by":2,"name":"Myoung Wha Hong","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Myoung","middleName":"Wha","lastName":"Hong","suffix":""},{"id":127805511,"identity":"e7b6654a-f59d-42eb-af35-bed4ba0d3ab4","order_by":3,"name":"Young Yul Kim","email":"","orcid":"","institution":"The Catholic University of Korea","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Young","middleName":"Yul","lastName":"Kim","suffix":""},{"id":127805512,"identity":"aabeb895-2b59-40aa-b019-4a88ae78615d","order_by":4,"name":"Young-Sam Cho","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYFADCQbGBwwHwExmorUwG5CshU2CKC0Gx3vMHn75YxfNP7v5WDXPmcMM/O0HmI0r8Gk5c8bcWLYtOXfGnWNpt3luHGaQOJPAnHgGn5YbOWbSkg3MuRskcsxu53w4zMBwg4H5YAMhLRJ/6sFaikFa5InRIvmB7TBYC3MO0GEGQC2J+LRInjlWJs3Ydjx3xo20ZOk/Z9J5DM8kNhvi08J3vHmb5I8/1bn9M5IPfpxxzFpO7vjhw5L4tCgcAEYDD5IAkM2ITwMDgzxQmvEHXiWjYBSMglEw4gEA35JR6FPZgrYAAAAASUVORK5CYII=","orcid":"","institution":"Wonkwang University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Young-Sam","middleName":"","lastName":"Cho","suffix":""}],"badges":[],"createdAt":"2022-07-28 05:14:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1903800/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1903800/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":25149285,"identity":"f4ef51c3-7b4d-44ad-84ef-cd8315dd87fb","added_by":"auto","created_at":"2022-08-12 16:23:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3048372,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic of the PCL/nHA/collagen scaffold with dual growth factors (BMP-2 and FGF-2): (a) 3D-printing system available to extrude PCL/nHA and collagen materials (lab-made extrusion type), (b) top and cross-sectional view of fabricated PCL/nHA/collagen scaffold with various collagen pattern.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1903800/v1/5700d3d2c1e2ee3f026b4d0f.png"},{"id":25149728,"identity":"affe6c90-cac5-4674-978e-b2b42914c2de","added_by":"auto","created_at":"2022-08-12 16:28:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1584171,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the chemical component, tensile modulus, and TGA results for prepared PCL/nHA scaffolds or PCL/nHA bulk with various nHA ratio: (a) FT-IR results for PCL and nHA pure materials, (b) FT-IR results for fabricated PCL/nHA scaffold, (c) TGA results for fabricated PCL/nHA scaffold [29], (d) tensile modulus for fabricated PCL/nHA bulks, (e) tensile modulus for fabricated PCL/nHA scaffolds [29] (NS: nonsignificant, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1903800/v1/c0a586c4eb64f251c5353dd5.png"},{"id":25148691,"identity":"c6c496a3-ec7d-43cc-94b2-8094bdefe861","added_by":"auto","created_at":"2022-08-12 16:18:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5516221,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro response of human osteoblast-like cell cultured on fabricated PCL/nHA scaffolds: (a) CCK-8 assay [29], (b) DNS contents [29], (c) Live \u0026amp; Dead stain at 14 days (NS: nonsignificant, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1903800/v1/47098e6753785226d2dcc2db.png"},{"id":25148694,"identity":"aebf7546-eaa5-4049-b845-9c1e9cea0d4a","added_by":"auto","created_at":"2022-08-12 16:18:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":660077,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro release profiles of BMP-2 and FGF-2 from the fabricated PCL/nHA/collagen scaffolds with various patterns\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1903800/v1/c02fa7a38de8e350ee9b1589.png"},{"id":25148693,"identity":"51d44a6c-d9ec-4a33-bddb-a97510f98bea","added_by":"auto","created_at":"2022-08-12 16:18:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2283896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cspan class=\"ql-cursor\"\u003e\u003c/span\u003eQuantitative analysis of bone formation in calvarial defect 8 weeks after implantation, as determined via micro-CT scanning: (a) calvarial defect model without scaffold, (b) PCL/nHA/collagen scaffold with positive patterns, (c) PCL/nHA/collagen scaffold with edge patterns, (d) PCL/nHA/collagen scaffold with radial patterns, (e) total bone volume at 8-mm ROI, (f) bone volume in the central area at 4-mm ROI (yellow arrows: defect point, *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1903800/v1/0d171650167202897ad9d499.png"},{"id":25148699,"identity":"a11e82a7-41a8-49e1-86eb-72618ae67dbb","added_by":"auto","created_at":"2022-08-12 16:18:51","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3753408,"visible":true,"origin":"","legend":"\u003cp\u003eH\u0026amp;E-stained images in the edge (A-A dot line: orange) and central areas (B-B dot line: black) of the calvarial defect 8 weeks after implantation: (a) calvarial defect model without scaffold, (b) PCL/nHA\u003cstrong\u003e/\u003c/strong\u003ecollagen scaffold with positive pattern, (c) PCL/nHA/collagen scaffold with edge pattern, (d) PCL/nHA/collagen scaffold with radial pattern (black arrows: defect point, S: scaffold).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1903800/v1/e14763510cfa53cb2b22b81a.png"},{"id":25149287,"identity":"2c163a47-3669-4d05-a8f7-a1f4a842d95e","added_by":"auto","created_at":"2022-08-12 16:23:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3909439,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Masson’s trichrome–stained images in the edge (A-A dot line: orange) and central areas (B-B dot line: black) of the calvarial defect 8 weeks after implantation: (a) calvarial defect model without scaffold, (b) PCL/nHA\u003cstrong\u003e/\u003c/strong\u003ecollagen scaffold with positive pattern, (c) PCL/nHA/collagen scaffold with edge pattern, (d) PCL/nHA/collagen scaffold with radial pattern (black arrows: defect point, S: scaffold).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-1903800/v1/d6019faf55f418a62664ca95.png"},{"id":25148695,"identity":"7c09680a-04d9-4dba-bb4d-2da26f3701c4","added_by":"auto","created_at":"2022-08-12 16:18:51","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3582883,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of IHC–stained images for CD31 antibody in the edge (A-A dot line: orange) and central areas (B-B dot line: black) of the calvarial defect 8 weeks after implantation: (a) calvarial defect model without scaffold, (b) PCL/nHA\u003cstrong\u003e/\u003c/strong\u003ecollagen scaffold with positive pattern, (c) PCL/nHA/collagen scaffold with edge pattern, (d) PCL/nHA/collagen scaffold with radial pattern (black arrows: defect point, S: scaffold).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-1903800/v1/488e948ac089b75e2e632607.png"},{"id":25148698,"identity":"a5a976ed-8bd7-4f36-bfa3-d02c1d5d5b0b","added_by":"auto","created_at":"2022-08-12 16:18:51","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":8127613,"visible":true,"origin":"","legend":"\u003cp\u003eHistological comparison of PCL/nHA/collagen scaffolds 8 weeks after implantation at high magnification (x100): (a) PCL/nHA/collagen scaffold with positive pattern, (b) PCL/nHA/collagen scaffold with edge pattern, PCL/nHA/collagen scaffold with radial pattern (c) (yellow arrows: newly formed blood vessel, S: scaffold).\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-1903800/v1/11e0b0f944aff8af47734790.png"},{"id":30675410,"identity":"65d375c4-a44b-4287-aef9-4ff20279b58f","added_by":"auto","created_at":"2022-12-22 14:29:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9110731,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1903800/v1/170d5194-0eec-4577-a6e0-721e20856bfd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Strategy to improve endogenous bone regeneration of 3D-printed polycaprolactone/hydroxyapatite composite scaffold: Collagen designs with bone morphogenetic protein 2 and fibroblast growth factor 2","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLarge bone defects with critical defect sizes may occur frequently due to traffic accidents, trauma, tumors (osteosarcoma), or congenital diseases in humans. In terms of bone regeneration, the endogenous regeneration of bone defects still represents a clinical challenge despite the development of intervention therapy to achieve bone regeneration via autologous grafts, allogeneic grafts, BMP-2, etc. [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The current intervention therapy using bone grafts may lead to drawbacks reported in the literature, such as the risk of disease transmission and immunogenic response, scarcity of bone donors, or discrepancy due to the limited shape of powder or block types [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In bone tissue engineering, a 3D synthetic scaffold with biocompatible and biodegradable properties, which has been developed as an alternative to conventional bone grafts, is regarded to play a key role along with cells and growth factor proteins associated with the improvement of bone cell differentiation and maturation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In the fabrication of a 3D synthetic scaffold, 3D printing has been highlighted because it can fabricate a customized 3D synthetic scaffold with a favorable design for target tissues and cells, including pore size, porosity, and geometrical characteristics, using computer-aided design/computer-aided manufacturing techniques (CAD/CAM) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany studies have focused on enhancing bone defect recovery using osteoconductive 3D synthetic scaffolds and osteoinductive growth factors. Osteoconduction refers to bone growth on the surface of a graft. Osteoinduction involves the recruitment of immature cells and stimulation of these cells to develop into preosteoblast cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In 3D-printing technique, to improve the osteoconduction ability and mechanical properties of 3D synthetic scaffolds, composite materials consisting of synthetic polymers (PCL, PLA, PVA, etc.) and bioceramics (HA, β-TCP, bioactive glass, etc.) have been reported in literature for bone regeneration [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To enhance the osteoinduction ability of 3D synthetic scaffolds, hybrid (synthetic polymer/ hydrogel) or hydrogel scaffolds loaded on bone morphogenetic proteins (BMPs) have been proposed [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. From the viewpoint of the endogenous regeneration of large bone defects, the formation of vascular networks by angiogenic growth factors has attracted attention as a strategy for large bone regeneration. The formation of a vascular network within the bone constructs could provide essential nutrients and oxygen via diffusion to the bone tissue or cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. To achieve endogenous regeneration of large bone defects, a 3D synthetic scaffold with dual growth factors consisting of BMPs and angiogenic (VEGF, fibroblast growth factor (FGF)-2, PDGF, etc.) growth factors has been reported in the literature [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. According to previous studies [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], the ratio, concentration, and type of BMPs and angiogenic growth factors influence the bone-regenerating ability of 3D synthetic scaffolds. We assumed that the 3D-printed collagen pattern with BMP-2 and FGF-2 growth factors in the 3D-printed 3D synthetic scaffold could be guided in the endogenous regeneration of bone defects. Therefore, in this study, to demonstrate our hypothesis, the PCL/nHA composite material with relatively enhanced osteoconduction and mechanical properties was used for the fabrication of 3D-printed 3D synthetic scaffolds. The kagome structure of the PCL/nHA scaffold was selected as a delivery system for growth factors because a scaffold with a kagome structure can be adjusted to have various collagen designs via an 3D-printing system. Moreover, the kagome structure can enhance the mechanical properties of the scaffold, including the compressive modulus, tensile modulus, and bending modulus, compared with those of the structure of a conventional scaffold fabricated using an 3D-printing system [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. PCL/nHA/collagen scaffolds with dual growth factors (BMP-2 and FGF-2) and various hydrogel patterns (positive, edge, and radial patterns) were fabricated at the same ratio and concentration of dual growth factors. The possibility of endogenous regeneration of bone defects was investigated using the aforementioned scaffold.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Preparation and fabrication of the PCL/nHA/collagen scaffold with dual growth factors (FGF-2 and BMP-2)\u003c/h2\u003e\n \u003cp\u003eCommercial PCL (M\u003csub\u003ew\u003c/sub\u003e= 43,000\u0026ndash;50,000 Da, Polysciences, Warrington, PA, USA) and nHA (particle size: \u0026lt; 200 nm, Sigma-Aldrich, St. Louis, MO, USA) were purchased to prepare the composite material. The PCL/nHA composite material (PCL/nHA) was prepared as described in our previous studies [\u003cspan class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. Briefly, PCL pellets were dissolved in 5% (w/v) dichloromethane (DCM; Daejung Chemicals and Materials, Siheung, Republic of Korea) using a magnetic stirrer for 1 h at 500 rpm. nHA powder (10 wt%) was added to a 5 w/v% PCL/DCM solution and dried in a vacuum oven at 80\u0026deg;C for 72 h. The dried PCL/nHA composite material was melted at 88\u0026deg;C for 1 h in a dispenser with the inner diameter of the dispenser ceramic nozzle being 100-\u0026micro;m. Simultaneously, to inject collagen with dual growth factors in the fabricating PCL/nHA scaffold with a kagome structure, 500 \u0026micro;L collagen type I (Concentration 3 w/v%) (COLTRIX\u0026reg;, Ubiosis, Seongnam, Republic of Korea), 50 \u0026micro;L BMP-2 solution of 2 \u0026micro;g/\u0026micro;L concentration (Recombinant Human/Murine/Rat BMP-2, PeproTECH, Rocky Hill, NJ, USA) solubilized in sterile water, and 50 \u0026micro;L FGF-2 solution of 1 \u0026micro;g/\u0026micro;L concentration (Recombinant Human FGF-basic, PeproTECH, Rocky Hill, NJ, USA) in 5 mM Tris buffer were carefully mixed by pipetting. The prepared collagen was injected into a 100 \u0026micro;L syringe (Gastight-type model 1705, Hamilton, Reno, NV, USA). Subsequently, the syringe was equipped with an 3D-printing system (lab-made extrusion type) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). As an early step in the fabrication process of the PCL/nHA/collagen scaffold, a 3D scaffold with a kagome structure was drawn via composite-material extrusion by rotating a single screw at 45 rpm under an air pressure of 250 kPa. Second, after the composite-material extrusion process, collagen droplets (0.5 \u0026micro;L or 1 \u0026micro;L) were injected into the pores of the 3D scaffold with a kagome structure according to design patterns, including positive, edge, and radial patterns, via the collagen-extruding system (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(a)). For the loading amount of BMP-2 and FGF-2 in the fabricated PCL/nHA/collagen scaffold, the loading amounts of each scaffold with various collagen patterns were calculated as 2-\u0026micro;g BMP-2 and 1-\u0026micro;g FGF-2. The parameters of the fabricated PCL/nHA/collagen scaffolds are presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Furthermore, the total volume of the injected collagen was adjusted to 13 \u0026micro;L for each scaffold with various collagen patterns. For gelation of the injected collagen in the fabricated scaffold, the PCL/nHA/collagen scaffold was placed in an incubator for 30 min at 37\u0026deg;C with 95% air and 5% CO\u003csub\u003e2\u003c/sub\u003e. In addition, to compare bone formation of the scaffold with various collagen patterns, PCL/nHA/collagen scaffolds with positive, edge, and radial patterns were fabricated (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(b)). In terms of the in vitro and in vivo experiments, the cubic-shape and plug-shape scaffolds with a dimension of 5 mm \u0026times; 5 mm \u0026times; 3.6 mm and D\u003csub\u003e1\u003c/sub\u003e:8.9 mm/D\u003csub\u003e2\u003c/sub\u003e:7.9 mm/H:1.3 mm were fabricated, respectively (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e(a)).\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDesign parameters of 3D-printed PCL/nHA/collagen scaffold\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" style=\"width: 22.9592%;\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 26.5306%;\"\u003e\n \u003cp\u003ePCL/nHA/collagen (positive pattern)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 24.8724%;\"\u003e\n \u003cp\u003ePCL/nHA/collagen (edge pattern)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" style=\"width: 25.6378%;\"\u003e\n \u003cp\u003ePCL/nHA/collagen (radial pattern)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 22.9592%;\"\u003e\n \u003cp\u003ePorosity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 26.5306%;\"\u003e\n \u003cp\u003e50.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 24.8724%;\"\u003e\n \u003cp\u003e49.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 25.6378%;\"\u003e\n \u003cp\u003e49.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 22.9592%;\"\u003e\n \u003cp\u003ePore size\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 26.5306%;\"\u003e\n \u003cp\u003e510\u0026thinsp;\u0026plusmn;\u0026thinsp;22 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 24.8724%;\"\u003e\n \u003cp\u003e509\u0026thinsp;\u0026plusmn;\u0026thinsp;17 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 25.6378%;\"\u003e\n \u003cp\u003e513\u0026thinsp;\u0026plusmn;\u0026thinsp;29 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 22.9592%;\"\u003e\n \u003cp\u003eCalculating total volume of collagen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 26.5306%;\"\u003e\n \u003cp\u003e13 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 24.8724%;\"\u003e\n \u003cp\u003e13 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 25.6378%;\"\u003e\n \u003cp\u003e13 \u0026micro;l\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 22.9592%;\"\u003e\n \u003cp\u003eCalculating amount of BMP-2 loaded\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 26.5306%;\"\u003e\n \u003cp\u003e2 \u0026micro;g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 24.8724%;\"\u003e\n \u003cp\u003e2 \u0026micro;g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 25.6378%;\"\u003e\n \u003cp\u003e2 \u0026micro;g\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" style=\"width: 22.9592%;\"\u003e\n \u003cp\u003eCalculating amount of FGF-2 loaded\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 26.5306%;\"\u003e\n \u003cp\u003e1 \u0026micro;g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 24.8724%;\"\u003e\n \u003cp\u003e1 \u0026micro;g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" style=\"width: 25.6378%;\"\u003e\n \u003cp\u003e1 \u0026micro;g\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Design of the PCL/nHA/collagen scaffold with kagome structure\u003c/h2\u003e\n \u003cp\u003eTo fabricate a PCL/nHA/collagen scaffold with a kagome structure using an 3D-printing system, the kagome structure was designed as reported in literature [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. The designed scaffold had the following structural characteristics: approximate porosity of 50% and pore size of 500 \u0026micro;m. The 3D-printing pathway based on the g-code was generated using slicing software (Slic3r, version 1.2.9) via the standard tessellation language file of the designed scaffold.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3 Scaffold characterization\u003c/h2\u003e\n \u003cp\u003eThe porosities of the PCL/nHA (10 wt%) scaffolds without collagen were calculated using Eq.\u0026nbsp;(\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) as follows (a total of 10 scaffolds were used):\u003c/p\u003e\n \u003cdiv class=\"Equation\" id=\"Equ1\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e$$\\text{Porosity }\\left(\\%\\right) = \\frac{{v}_{1}-\\left(\\frac{\\text{0.9}\\times m}{{\\rho }_{\\text{PCL}}}+\\frac{0.1\\times m}{{\\rho }_{\\text{HA}}}\\right)}{{v}_{1}}\\times 100$$\u003c/div\u003e\n \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({v}_{1}\\)\u003c/span\u003e\u003c/span\u003e is the calculated apparent volume of the fabricated PCL/nHA scaffold via the scaffold\u0026rsquo;s outer dimension, \u003cspan style='color: rgb(0, 0, 0); font-family: \"Times New Roman\"; font-size: medium; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: 400; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; text-decoration-thickness: initial; text-decoration-style: initial; text-decoration-color: initial; display: inline !important; float: none;'\u003e\u003cem\u003e\u0026rho;\u003c/em\u003e\u003c/span\u003e\u003cem\u003e\u003c/em\u003e\u003csub\u003ePCL\u003c/sub\u003e and \u003cem\u003e\u0026rho;\u003c/em\u003e\u003csub\u003eHA\u003c/sub\u003e are the densities of PCL and nHA, respectively, and m is the weight of the fabricated scaffold. To compare the pore size of the fabricated PCL/nHA scaffold without collagen, the vertical and horizontal distances of the apparent pores on the surface of the scaffold were averaged using an optical microscope (MI-9100 ZOOM, Magic i, Seoul, Republic of Korea). Repetitive experiments were performed for each type via 10 scaffolds.\u003c/p\u003e\n \u003cp\u003eChemical analysis of the pure PCL, nHA, and PCL/nHA scaffolds with various nHA weight ratios (3, 5, and 10 wt%) was performed using Fourier transform infrared spectroscopy (FT-IR; 6300FV, JASCO, Tokyo, Japan). Repetitive experiments were performed for each sample type using two samples. Moreover, the mass of the fabricated scaffold\u0026rsquo;s residual material from 30 to 600\u0026deg;C was investigated using TGA (SDT Q600, TA instruments, New Castle, DE, USA). Three scaffolds were used for each type. To determine the tensile modulus of PCL/nHA bulk with various nHA weight ratios (3, 5, and 10 wt %) and PCL/nHA scaffolds with various nHA weight ratios (3, 5, and 10 wt%), tensile tests were conducted (UTM; MTS, Eden Prairie, MN, USA) at a constant strain rate of 1 mm/ min with a 5 kN loading cell. For the tensile test, dumbbell-shaped composite bulk and PCL/nHA scaffolds were fabricated according to the ISO standard (KS M ISO 527-4:2002). The tensile moduli of the PCL/nHA bulk and PCL/nHA scaffold were determined in the linear region within 1% strain of the stress-strain (S-S) curve. Repetitive experiments were performed for each sample type using eight samples.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4 Release profile of BMP-2 and FGF-2\u003c/h2\u003e\n \u003cp\u003eThe release of BMP-2 and FGF-2 from the prepared PCL/nHA/collagen scaffold in the culture medium was analyzed by ELISA (R\u0026amp;D Systems, Minneapolis, MN, USA). The prepared PCL/nHA/collagen scaffold was placed in 2 ml Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM; Gibco, New York, NY, USA), and supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 \u0026micro;g/mL streptomycin, and 100 U/ mL penicillin (Gibco, Carlsbad, CA, USA). The cells were maintained in an incubator at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The culture medium was collected for 3 weeks. The concentrations of released BMP-2 and FGF-2 growth factors were calculated according to the ELISA kit protocols.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5 Human osteoblast-like cell culture and proliferation\u003c/h2\u003e\n \u003cp\u003eCultured human osteogenic sarcoma cells after a few passages (Saos-2 cells; Korea Cell Line Bank, Seoul, Republic of Korea) were cultured in DMEM (Gibco, New York, NY, USA), and supplemented with 10% FBS (Gibco, Grand Island, NY, USA), 100 \u0026micro;g/mL streptomycin, and 100 U/mL penicillin (Gibco, Carlsbad, CA, USA) in a humidified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The prepared PCL/nHA scaffolds were sterilized by immersion in 70% EtOH overnight under UV light. The cells in suspension (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/scaffold) were seeded onto the surface of the prepared PCL/nHA scaffolds. Cultures of cell-seeded scaffolds were maintained at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. To evaluate cell adhesion and proliferation in the scaffold, qualitative cell numbers were determined using a cell counting kit (CCK-8; Dojindo, Kumamoto, Japan) at an absorbance of 450 nm using a multimode plate reader after culturing for 1, 7, and 14 days. The viability of human osteoblast-like cells on the scaffolds was investigated using a live/dead kit (Molecular Probes, Eugene, OR, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6 Scaffold implantation without cells in rat\u0026rsquo;s calvarial defect for in vivo study\u003c/h2\u003e\n \u003cp\u003eThe in vivo study was performed according to all procedures and principles approved by the Institutional Review Board of St. Mary\u0026rsquo;s Hospital of Catholic University (CMCDJ-AP-2019-005). To anesthetize eight-week-old SD rats ( n\u0026thinsp;=\u0026thinsp;5), intraperitoneal injections of ketamine hydrochloride (Yuhan, Seoul, Republic of Korea) and Rompun\u0026reg; (Bayer, Leverkusen, Germany) were administered. After anesthetized SD rats were placed on a heated pad, the surgical site of SD rats was shaved and sterilized with povidone. The rats were placed on an operating table with a heated pad and covered with a sterile drape. After a longitudinal midline incision was made, the periosteum and skin were carefully detached. To create a circular defect with a diameter of 8-mm in the calvarial bone, an 8-mm trephine bur was utilized. Each PCL/nHA/collagen scaffold with positive, edge, and radial patterns was implanted into a calvarial defect. To finish the scaffold implantation, the cleaved skin was sutured with 4\u0026thinsp;\u0026minus;\u0026thinsp;0 black silk (Ailee, Busan, Republic of Korea). To investigate bone formation by scaffold implantation in the calvarial defect, SD rats were sacrificed in the eighth week after implantation. Calvarial samples were harvested from sacrificed SD rats. The cells were then fixed with 10% formalin. All the surgical procedures were performed under aseptic conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.7 Quantitative analysis of the bone formation by PCL/nHA/collagen scaffold via \u0026micro;CT\u003c/h2\u003e\n \u003cp\u003eTo quantitatively analyze bone formation in the scaffolds, the harvested calvarial samples were scanned using a micro-CT (\u0026micro;CT) scanner (Skyscan 1172, Bruker, Billerica, MA, Belgium). The harvested calvarial samples were scanned (resolution: 13 \u0026micro;m and aluminum filter: 500 \u0026micro;m), and micro-radiographic images were obtained at 60 kV and 167 \u0026micro;A. To reconstruct the harvested calvarial samples, the 6-bit scanning files obtained were converted into DICOM files (DicomCT version 2.5, Bruker, Billerica, MA, USA). The harvested calvarial samples were graphicalized using prepared DICOM files and reconstruction software (Mimics version 21.0, Materialise, Leuven, Belgium). For this 3D remodeling, the threshold values (lower and upper) for natural bone were presumed to be 95 and 250 Hounsfield units. The total bone volume within the cylindrical region of interest (ROI; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(8\\times 1\\text{ mm}\\)\u003c/span\u003e\u003c/span\u003e or \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(4\\times 1\\text{ mm}\\)\u003c/span\u003e\u003c/span\u003e) was measured by assigning an assumed threshold value for the total bone content. Five harvested calvarial samples were analyzed in each group. All scanning and 3D remodeling parameters were identical in all the groups. Moreover, all \u0026micro;CT images were selected that were similar to the average values of the measured bone volume.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e2.8 Preparation for histological and immunohistochemistry (IHC) staining\u003c/h2\u003e\n \u003cp\u003eSpecimens for histological and immunohistochemical analyses were prepared after scanning harvested calvarial samples. Fixation and decalcification of harvested calvarial samples were performed by immersion in 10% formalin and 10% nitric acid solution. After fixation and decalcification, calvarial samples were embedded in paraffin. The calvarial samples were embedded in paraffin. Thereafter, the slides of calvarial samples were stained with hematoxylin and eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome stains after the paraffin blocks were cut to 4-\u0026micro;m thicknesses. Masson\u0026rsquo;s trichrome staining was performed according to the manual of the Masson\u0026rsquo;s trichrome stain kit (Polysciences, Warrington, PA, USA). For immunohistochemical staining, paraffin tissue slides were dissolved in xylene to remove the paraffin. The deparaffinized tissue slides were rehydrated using serial concentrations of ethanol. After washing, rehydrated tissue slides were retrieved using citrate buffer. Thereafter, the tissue slides were blocked for 30 min and incubated with a rat anti-CD31 antibody (Novus biologicals, Littleton, CO, USA) overnight at 4\u0026deg;C. The tissue slides were incubated with secondary antibodies for 1 h and observed using an ImmPACT\u003csup\u003e\u0026reg;\u003c/sup\u003e NovaRED\u003csup\u003e\u0026reg;\u003c/sup\u003e substrate peroxidase (SK-4805; Vector Laboratories, Burlingame, CA, USA).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eStatistical analysis was performed via Student\u0026rsquo;s t-test (Microsoft 2019, Redmond, WA, USA) using the measured data. Student\u0026rsquo;s t-test was performed considering a value of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The obtained data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003ch2\u003e3.1 Evaluation of in vitro characteristics for the fabricated PCL/nHA scaffolds without collagen at a specific ratio of nHA\u003c/h2\u003e\n\u003cp\u003eTo compare the characteristics of the fabricated PCL/nHA scaffolds with a kagome structure, the HA weight ratio, tensile modulus, and in vitro cell response were investigated. The nHA amounts in the fabricated PCL/nHA scaffolds were determined via FT-IR and TGA before evaluating the characteristics of the fabricated PCL/nHA scaffolds. We revealed that the detection level of the P-O group, which is a representative chemical component of nHA, increased with the nHA weight ratio of the fabricated scaffolds (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(a) and (b)). Moreover, the PCL and PCL/nHA (3, 5, and 10 wt%) scaffolds\u0026rsquo; residuals at 600\u0026deg;C were measured as 0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1%, 3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6%, 5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7%, and 10.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2% (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e(c)). We verified that the actual nHA amount in the fabricated scaffolds was similar to the designed HA weight ratio of the PCL/nHA scaffold. The tensile moduli of PCL and PCL/nHA (3, 5, and 10 wt%) bulks were 327.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 MPa, 334.5\u0026thinsp;\u0026plusmn;\u0026thinsp;12.1 MPa, 340.0\u0026thinsp;\u0026plusmn;\u0026thinsp;7.7 MPa, and 398.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.6 MPa, respectively. The tensile moduli of PCL and PCL/nHA (3, 5, and 10 wt%) scaffolds were found to be 86.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 MPa, 97.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2 MPa, 106.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 MPa, and 121.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.8 MPa, respectively. In terms of in vitro osteoblast-like cell response, the proliferation and viability of cells cultured on fabricated PCL/nHA scaffolds for 2 weeks were investigated at the nHA\u0026rsquo;s specific ratio (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The cell proliferation of the PCL/nHA scaffold with 10 wt% nHA was higher than that of other scaffolds, although there was no difference in the cell adhesion of the fabricated PCL/nHA scaffolds with increasing nHA weight ratio (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e(a) and (b)). Moreover, the numbers of dead cells on the surface of the PCL/nHA scaffold with 10 wt% were less than that on the other scaffolds.\u003c/p\u003e\n\u003ch2\u003e3.2 Assessment of release profiles of loaded dual growth factors and bone formation of PCL/nHA/collagen scaffolds with various collagen patterns\u003c/h2\u003e\n\u003cp\u003eThe release trends of dual growth factors (BMP-2 and FGF-2) loaded on the PCL/nHA/collagen scaffolds were investigated according to the proposed collagen patterns (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). In terms of BMP-2 release (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e(a)), the release trend of BMP-2 from the PCL/nHA/collagen scaffold with a positive pattern was similar to the radial pattern over 21 days. Although BMP-2 release from the PCL/nHA/collagen scaffold with edge pattern was slightly different compared with the positive and radial patterns before 7 days, the released concentration of the PCL/nHA/collagen scaffold with edge pattern was similar to that of other scaffolds after 14 days. Furthermore, the release trends of FGF-2 from all groups were virtually the same over 21 days (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e(b)). Consequently, the total concentrations of dual growth factors released from the prepared scaffolds with various collagen patterns were similar. For quantitative analysis of bone formation via micro-CT (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), the average bone volumes in accordance with 8-mm ROI in the calvarial defect model without scaffold and the PCL/nHA/collagen scaffold with various patterns (positive, edge, and radial patterns) were measured as 4.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 mm\u003csup\u003e3\u003c/sup\u003e, 15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.4 mm\u003csup\u003e3\u003c/sup\u003e, 14.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.2 mm\u003csup\u003e3\u003c/sup\u003e, and 27.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 mm\u003csup\u003e3\u003c/sup\u003e, respectively. The bone-regenerating levels of all the groups for 4-mm ROI were investigated as 0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 mm\u003csup\u003e3\u003c/sup\u003e, 0.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mm\u003csup\u003e3\u003c/sup\u003e, 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 mm\u003csup\u003e3\u003c/sup\u003e, and 5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4 mm\u003csup\u003e3\u003c/sup\u003e, respectively. The bone-regenerating level of all scaffolds was superior to that of the calvarial defect model without scaffold at 8 weeks after implantation. Furthermore, we revealed that the bone volumes were different between scaffold groups based on collagen patterns with dual growth factors at the same concentration and total volume loaded with collagen.\u003c/p\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003e3.3 Histological evaluation of bone formation and angiogenesis in the fabricated scaffolds\u003c/h2\u003e\n \u003cp\u003eHistological and IHC analyses for newly formed bone and blood vessels were performed using H\u0026amp;E, Masson\u0026rsquo;s trichrome, and IHC staining for CD31 antibody at 8 weeks after implantation (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). For H\u0026amp;E and Masson\u0026rsquo;s trichrome staining, the tissues in the calvarial defect model without the scaffold were relatively loose (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). In contrast, matured bone tissues were observed in all scaffold groups compared to that seen in the calvarial defect model without scaffold (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e, and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). Furthermore, the matured bone tissues in the PCL/nHA/collagen scaffolds with a radial pattern were higher than those in the other scaffolds. For immunohistochemical staining, formation of numerous blood vessels in scaffold groups was identified compared with that seen in the calvarial defect model without scaffold (Figs. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). The formation of blood vessels in the mature bone tissue of the PCL/nHA/collagen scaffold with a radial pattern was greater than that of the other scaffolds (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). The action level of the CD 31 antibody between the scaffold groups was similar.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTo investigate the influence of various collagen patterns loaded with dual growth factors (BMP-2 and FGF-2) in the implanted PCL/nHA/collagen scaffold on endogenous bone formation and angiogenesis, the pore size, porosity, concentration of dual growth factors, and total volume of loaded collagen were fixed for each type, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. In terms of pore size and porosity, an interconnected pore size of 300\u0026ndash;500 \u0026micro;m and 50% porosity are recommended to promote bone formation and vascularization in the 3D scaffold reported in the literature [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, the pore size and porosity of the scaffold with a kagome structure were fixed at 500 \u0026micro;m and 50%, respectively, to exclude the effect of the geometric parameters of the scaffold on the bone-regenerating ability. For the weight ratio of the PCL/nHA composite material, 10 wt% of nHA was considered as the maximum weight ratio because the composite material with over 10 wt% nHA could have disadvantages, including non-uniform extrusion and nozzle clogging. Regarding the concentration of BMP-2 and FGF-2, BMP-2 has been known to have an important role in osteogenesis; for example, it is involved in the differentiation of mesenchymal stem cells to osteoblasts or chondrocytes for bone recovery [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. FGF-2 can promote angiogenesis. Generally, angiogenesis is considered an essential process for recovery from damaged bone. Moreover, newly formed blood vessels can provide nutrients, growth factors, and stem cells to the damaged bone site [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, we selected BMP-2 and FGF-2 as the growth factors for bone recovery. From the viewpoint of the amount and concentration of dual growth factors, Kim et. al. reported that bone formation induced by BMP-2 did not increase when the injection amount exceeded 1-\u0026micro;g in a mouse calvarial defect model [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, Liu et. al. reported that the bone-regenerating activity of a 2:1 weight ratio (BMP-2:FGF-2) was superior to other weight ratios for BMP-2 and FGF-2 (1:1, 2:1, 4:1, and 8:1) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Tabata et. al. verified the synergistic effect of BMP-2/FGF-2 (2:1 weight ratio) and BMP-2/VEGF (3:1 weight ratio), which are known to be suitable weight ratios of dual growth factors for bone regeneration [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The synergistic effect of BMP-2/FGF-2 (2:1 weight ratio) was better than that of BMP-2/VEGF (3:1 weight ratio) in bone regeneration and vascular formation. In this study, the BMP-2 amount loaded onto the scaffold with a kagome structure was fixed at 2 \u0026micro;g. Furthermore, to maximize the synergistic effect of BMP-2 and FGF-2, the weight ratio was fixed at 2:1 (BMP-2:FGF-2). For the hydrogel material, the collagen type I was selected as a carrier system of the dual growth factors because bone tissues are primarily composed of 70% mineral (primarily hydroxyapatite), 22% protein (primarily collagen type I), and 8% other components [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the in vitro experiment, a change in the mechanical properties of a composite bulk/3D-printed scaffold and the human osteoblast-like cell response of the 3D scaffold were investigated according to the increasing weight ratio of nHA in the PCL/nHA composite material before the in vivo experiment using a rat calvarial defect model. To verify the actual weight ratio of the nHA powder in the prepared PCL/nHA composite material, changes in the chemical components and amount of residual material at 600\u0026deg;C were measured because PCL was incinerated at approximately 450\u0026deg;C (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a-c)). The detection level of the P-O group increased with the nHA weight ratio in the PCL/nHA scaffolds. In contrast, the P-O group was not detected in the pure PCL and PCL scaffolds (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a) and (b)). Moreover, the actual amount of nHA powder was similar to the designed weight ratio of nHA powder in the PCL/nHA composite material (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c)). With respect to the tensile modulus of bulk and 3D-printed scaffolds, the tensile modulus of the PCL bulk was similar to that of the PCL/nHA bulk (3 and 5 wt%). However, the tensile modulus of the PCL/nHA bulk (10 wt%) was higher than that of the other bulk materials (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(d)). The tensile modulus of the PCL/nHA scaffold was enhanced by increasing the nHA weight ratio. Specifically, the tensile modulus of the PCL/nHA scaffold (10 wt. %) was superior to that of the other scaffolds. Moreover, with respect to the response of human osteoblast-like cells, the cell-adhesion abilities of the PCL and PCL/nHA scaffolds with various weight ratios of nHA did not differ significantly (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This phenomenon could explain why the weight ratio of nHA in the PCL/nHA composite material did not influence cell adhesion. However, the cell growth abilities of PCL/nHA scaffolds with various weight ratios were higher than those of PCL scaffolds at 7 and 14 days after cell culture on the fabricated scaffold because of the osteoconductive property of nHA. Furthermore, the cell growth level of the PCL/nHA scaffold with 10 wt% nHA was superior to that of other scaffolds after 7 days (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a) and (b)). The number of dead cells on the PCL/nHA scaffold with 10 wt% nHA was less than that on the other scaffolds (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(c)). Consequently, to investigate endogenous bone regeneration and angiogenesis by various collagen design patterns with dual growth factors, the PCL/nHA scaffold with 10 wt% nHA was selected because it has enhanced mechanical properties and higher cell response compared to that of other scaffolds. Additionally, to compare the release trends of dual growth factors from the PCL/nHA/collagen scaffolds with various patterns, cumulative release was measured over 21 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). For the release profile of BMP-2, although the concentration of BMP-2 released from PCL/nHA/collagen with an edge pattern was different compared to the positive and radial patterns, the released concentrations of BMP-2 from all scaffolds were saturated after 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a)). Furthermore, the overall trends of released FGF-2 from all scaffolds were similar for 21 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(b)). This result indicated that the proposed collagen patterns did not affect the release profile of the dual growth factors.\u003c/p\u003e \u003cp\u003eFor the in vivo experiment using the rat calvarial defect model, to assess endogenous bone regeneration and angiogenesis, the analysis area was divided from the edge and central areas (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). From the micro-CT results, the bone volumes of all scaffolds in the 4-mm and 8-mm ROI were superior to those of the calvarial defect model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(e)). The bone formation level of the PCL/nHA/collagen scaffold with a radial pattern in the 8-mm ROI was higher than those of other scaffolds. Specifically, the bone volume of the PCL/nHA/collagen scaffold with a radial pattern in the 4-mm ROI was significantly higher than that of the other scaffolds. This phenomenon could explain why the existence or nonexistence of space for endogenous bone regeneration, along with dual growth factors, affected bone regeneration. In other words, a collagen design loaded with dual growth factors in the scaffold could affect endogenous bone regeneration. For the H\u0026amp;E- and Masson\u0026rsquo;s trichrome\u0026ndash;stained sections, matured bone tissue in all scaffolds was strongly expressed compared to that in the calvarial defect model (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In the edge area, many bone tissues were newly formed in all scaffolds. However, in the central area, mature bone tissue in the PCL/nHA/collagen scaffold with a radial pattern was more abundant than that in the other scaffolds (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). This result was in accordance with the micro-CT results. For IHC and staining, the expression levels of endothelial cells in all scaffolds were higher than those in the calvarial defect model (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). The expression levels of endothelial cells did not differ between the scaffold groups. However, many blood vessels in the newly formed tissues of the PCL/nHA/collagen scaffold with a radial pattern were already observed compared to that seen with other scaffolds (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e(c)). Consequently, we demonstrated that a collagen design loaded with dual growth factors in the 3D-printed scaffold could affect endogenous bone regeneration and angiogenesis. Therefore, the provision of space for bone regeneration must be considered seriously when collagen design contains a dual growth factor in the 3D-printed scaffold. In our opinion, the clinical problem of endogenous regeneration of bone defects can be overcome by the 3D-printed scaffold in consideration of the abovementioned design parameters.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, to evaluate the influence of collagen patterns with dual growth factors in the PCL/HA scaffold on endogenous bone regeneration and angiogenesis, PCL/nHA/collagen scaffolds with kagome structure and various collagen patterns (positive, edge, and radial patterns) were fabricated using an 3D-printing system (material extrusion type). In terms of nHA weight ratio, the mechanical properties and human osteoblast-like cell response of the PCL/nHA scaffold with 10 wt% nHA were superior to those of the other scaffolds. We found that the in vitro released concentrations of BMP-2 and FGF-2 were not affected by collagen patterns in the PCL/nHA scaffold with 10 wt% nHA. Furthermore, endogenous bone regeneration and angiogenesis in the PCL/nHA/collagen scaffold with a radial pattern were promoted compared with those in the PCL/nHA/collagen scaffolds with positive and edge patterns. Therefore, we demonstrated that a collagen design loaded with dual growth factors in the 3D-printed scaffold could affect endogenous bone regeneration and angiogenesis. In other words, a design that considers the supply of space for bone regeneration is important when collagen design incorporates dual growth factors in the 3D-printed scaffold for effective endogenous regeneration in bone tissue engineering.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2021R1A2C2009665).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Yong Sang Cho and Young-Sam Cho, methodology, Yong Sang Cho, Myoung Wha Hong, and Young Yul Kim, software, Yong Sang Cho and Min-Soo Ghim, writing-original draft preparation, Yong Sang Cho, Min-Soo Ghim, and Myoung Wha Hong, writing-review and editing, Yong Sang Cho and Young-Sam Cho, supervision, Young Yul Kim and Young-Sam Cho.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePerier-Metz C, Duda GN, Checa S (2020) Mechano-Biological Computer Model of Scaffold-Supported Bone Regeneration: Effect of Bone Graft and Scaffold Structure on Large Bone Defect Tissue Patterning. 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Bioact Mater 6:3254\u0026ndash;3268\u003c/li\u003e\n\u003cli\u003eSantos MI, Reis RL (2010) Vascularization in bone tissue engineering: Physiology, current strategies, major hurdles and future challenges. Macromol Biosci 10:12\u0026ndash;27\u003c/li\u003e\n\u003cli\u003eKoria P (2012) Delivery of growth factors for tissue regeneration and wound healing. BioDrugs 26:163\u0026ndash;175\u003c/li\u003e\n\u003cli\u003eHajimiri M, Shahverdi S, Kamalinia G, Dinarvand R (2015) Growth factor conjugation: Strategies and applications. J Biomed Mater Res Part A 103:819\u0026ndash;838\u003c/li\u003e\n\u003cli\u003ePark JY, Shim JH, Choi SA, Jang J, Kim M, Lee SH et al (2015) 3D printing technology to control BMP-2 and VEGF delivery spatially and temporally to promote large-volume bone regeneration. J Mater Chem B 3:5415\u0026ndash;5425\u003c/li\u003e\n\u003cli\u003eCai WX, Zheng LW, Li CL, Ma L, Ehrbar M, Weber FE et al (2014) Effect of different rhBMP-2 and TG-VEGF ratios on the formation of heterotopic bone and neovessels. Biomed Res Int 2014:571510\u003c/li\u003e\n\u003cli\u003eCharles LF, Woodman JL, Ueno D, Gronowicz G, Hurley MM, Kuhn LT (2015) Effects of low dose FGF-2 and BMP-2 on healing of calvarial defects in old mice. Exp Gerontol 64:62\u0026ndash;69\u003c/li\u003e\n\u003cli\u003eLee SH, Cho YS, Hong MW, Lee BK, Park Y, Park SH et al (2017) Mechanical properties and cell-culture characteristics of a polycaprolactone kagome-structure scaffold fabricated by a precision extruding deposition system. Biomed Mater 12:055003\u003c/li\u003e\n\u003cli\u003eCho YS, Quan M, Kang NU, Jeong HJ, Hong MW, Kim YY et al (2020) Strategy for enhancing mechanical properties and bone regeneration of 3D polycaprolactone kagome scaffold: Nano hydroxyapatite composite and its exposure. 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J Biomed Mater Res Part B Appl Biomater 105:904\u0026ndash;925\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bone tissue engineering, 3D printing, Polymer-matrix composites, Collagen, Hybrid scaffold.","lastPublishedDoi":"10.21203/rs.3.rs-1903800/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1903800/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn bone tissue engineering, the endogenous regeneration of bone defects still represents a clinical challenge despite the development of intervention therapy to achieve bone regeneration via autologous grafts, allogeneic grafts, bone morphogenetic protein (BMP)-2, etc. To overcome the limitation of endogenous bone regeneration, we assumed that the 3D-printed collagen pattern with BMP-2 and fibroblast growth factor (FGF)-2 in the 3D-printed polycaprolactone/nano-hydroxyapatite scaffold could guide the endogenous regeneration of bone defects. Therefore, to test our hypothesis, polycaprolactone/nano-hydroxyapatite/collagen scaffolds with dual growth factors (BMP-2 and FGF-2) and various hydrogel patterns (positive, edge, and radial patterns) were fabricated at the same ratio and concentration of dual growth factors. Consequently, we revealed that the in vitro released concentrations of BMP-2 and FGF-2 were not affected by collagen patterns in the PCL/nano-hydroxyapatite scaffold. Furthermore, endogenous bone regeneration and angiogenesis in the polycaprolactone/nano-hydroxyapatite/collagen scaffold with a radial pattern were promoted compared with those in the polycaprolactone/nano-hydroxyapatite/collagen scaffolds with positive and edge patterns. Therefore, we demonstrated that a collagen design loaded with dual growth factors in the 3D-printed scaffold could affect endogenous bone regeneration and angiogenesis.\u003c/p\u003e","manuscriptTitle":"Strategy to improve endogenous bone regeneration of 3D-printed polycaprolactone/hydroxyapatite composite scaffold: Collagen designs with bone morphogenetic protein 2 and fibroblast growth factor 2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-08-12 16:18:48","doi":"10.21203/rs.3.rs-1903800/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1a508011-a5d8-4db0-ac72-2346f48fe012","owner":[],"postedDate":"August 12th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-12-22T14:29:33+00:00","versionOfRecord":[],"versionCreatedAt":"2022-08-12 16:18:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1903800","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1903800","identity":"rs-1903800","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}