Abstract

   The study focused on the effects of a triply periodic minimal surface
   (TPMS) scaffolds, varying in porosity, on the repair of mandibular
   defects in New Zealand white rabbits. Four TPMS configurations (40%,
   50%, 60%, and 70% porosity) were fabricated with β-tricalcium phosphate
   bioceramic via additive manufacturing. Scaffold properties were
   assessed through scanning electron microscopy and mechanical testing.
   For proliferation and adhesion assays, mouse bone marrow stem cells
   (BMSCs) were cultured on these scaffolds. In vivo, the scaffolds were
   implanted into rabbit mandibular defects for 2 months. Histological
   staining evaluated osteogenic potential. Moreover, RNA-sequencing
   analysis and RT-qPCR revealed the significant involvement of
   angiogenesis-related factors and Hippo signaling pathway in influencing
   BMSCs behavior. Notably, the 70% porosity TPMS scaffold exhibited
   optimal compressive strength, superior cell proliferation, adhesion,
   and significantly enhanced osteogenesis and angiogenesis. These
   findings underscore the substantial potential of 70% porosity TPMS
   scaffolds in effectively promoting bone regeneration within mandibular
   defects.

   Keywords: TPMS bone scaffold, osteogenesis, mandibular defect, bone
   regeneration, 3D printer/additive manufacture

Graphical abstract

   graphic file with name 10.1177_20417314241244997-img1.jpg

Introduction

   The maxillofacial regions, including the maxilla and nasal bones, serve
   as crucial supporting structures for maintaining facial contours and
   performing functions such as speech and mastication.^ [45]1 Bone
   defects in the maxillofacial region caused by tumor resection, trauma,
   and osteonecrosis^ [46]2 (radiogenic osteonecrosis and drug-related
   osteonecrosis) can significantly impair patients’ appearance, causing
   restricted speech and difficulty in eating, bringing substantial
   psychological and physiological burden to the patients.^ [47]3 Among
   the maxillofacial bones, the mandible is prone to various cysts and
   tumors,^ [48]4 superficially located and vulnerable to traumatic
   injury,^ [49]5 and possesses a singular blood supply making it
   susceptible to osteonecrosis^ [50]6 and osteomyelitis.^ [51]7
   Therefore, the repair and functional reconstruction of mandibular
   defects has long been a focus in the field of oral and maxillofacial
   surgery as well as plastic surgery,^ [52]8 bearing high clinical value
   and scientific significance.

   Mandibular defects can be categorized into small bone defects that can
   self-heal and critical-sized bone defects (CSBD) that cannot fully
   regenerate naturally. CSBD is clinically defined as defects exceeding
   50% of the bone circumference or greater than 2 cm. These defects are
   required surgical intervention using bone graft methods.^ [53]9
   Currently, bone graft materials are mainly derived from autogenous,
   allogeneic, and xenogeneic tissue sources.^ [54]10 Autografts are the
   “gold standard” source for bone grafting,^ [55]11 but their clinical
   application is limited due to restrictions on bone harvesting,
   “secondary surgery” trauma, and high incidence of complications at the
   donor site (8.5%–20%^ [56]12 ). Allograft and xenografts pose risks
   such as surgical complexity, high cost, and risk of pathogenic
   microbial infection.^ [57]13 Bone tissue engineering uses artificially
   synthesized, biologically active biomaterials to achieve good bone
   conduction, bone generation, and osteogenesis.^ [58]14 It is considered
   a potential treatment method to address the clinical limitations of
   existing treatment approaches.

   Currently, calcium phosphate (CAP) ceramics, such as hydroxyapatite
   (HA) and β-tricalcium phosphate (β-TCP), are commonly used as bone
   graft substitutes in the clinic. They share a similar crystalline
   structure with natural cancellous bone,^ [59]15 both exhibiting good
   bone induction and osteogenic activity.^ [60]16 Moreover, these
   materials can completely biodegrade in the body, with their degradation
   products Ca^2+ and PO[4]^ [61]3 − being the main components of the bone
   matrix, which promote bone mineralization.^ [62]17 HA requires several
   years to fully degrade in the body,^ [63]18 a slow degradation rate
   that is not synchronized with the body’s bone regeneration speed. In
   contrast, β-TCP can fully degrade in a matter of months,^ [64]19
   facilitating the dynamic balance of scaffold degradation and tissue
   regeneration,^ [65]20 thus making it become a more suitable material as
   bone tissue engineering scaffold. Bone tissue can be divided into two
   types: cortical bone (also known as dense bone) and trabecular bone
   (also known as cancellous bone or spongy bone). The difference between
   these two types of bone tissues is largely based on porosity: with the
   cortical bone possessing a porosity of 5% to 15%, while the porosity of
   trabecular bone is between 40% and 95%.^ [66]21 On the basis of
   scaffold materials simulating natural bone, the morphology of the bone
   scaffold should also adopt a structure similar to human trabecular
   bone. Trabecular bone is an internally interconnected three-dimensional
   porous structure, with pore diameters ranging from 100 to 900 μm, and
   the average curvature of the trabecular surface is zero.^ [67]22
   Current bone scaffolds often use a log-pile structure, but their flat
   surfaces and sharp edges, compared to the curved and smooth cellular
   partitions in the body, are not conducive to cell recruitment,
   adhesion, and growth.^ [68]23 It has been reported that triply periodic
   minimal surfaces (TPMS) with periodic changes along the X, Y, and Z
   axes in Euclidean space and it has a zero average curvature,^ [69]24
   which recapitulates the natural structure of cancellous bone.^ [70]25
   TPMS can be divided into Gyroid (G), Diamond (D), and Schwarz P (P)
   surfaces.^ [71]26 Among them, the G surface, due to its relatively
   larger specific surface area and porosity, facilitates cellular
   recruitment and promotes cell adhesion,^ [72]27 in addition to
   exhibiting characteristics that enhance osteogenic differentiation in
   the body.^ [73]28

   In this present study, we investigated the use of TPMS-G surface
   scaffolds, adjusting the function period to control pore size,
   combining internal offset, external offset, and triangles to precisely
   control scaffold wall thickness and porosity. We determined the
   influence of augmenting these parameters on important scaffold
   characteristics such as elastic modulus, compressive strength, and
   permeability parameters, in addition to evaluating its regenerative
   capacity in vitro and in vivo within a mandibular defect model in
   rabbits.

Materials and methods

Design and preparation of scaffold

   Using Matlab R2020a software, four G-surface scaffolds with porosities
   of 40%, 50%, 60%, and 70% were designed. The scaffolds were designed to
   be theoretically cylindrical, with a diameter of 8 mm, a height of
   4 mm, and a wall thickness of 0.25 mm. The scaffolds were created as
   cylinders with a bottom diameter of 8 mm, a thickness of 4 mm, and a
   wall thickness of 200 µm. β-tricalcium phosphate (β-TCP) scaffold was
   prepared by additive manufacturing technology based on digital laser
   processing. The bone scaffold we prepared contains 85% of light cured
   monomers β- Triple cycle minimum curved porous ceramic scaffold with
   tricalcium phosphate and 15% hydroxyapatite. The process parameters for
   3D printing (AUTOCERA-M, Beijing Shiwei Technology Co. Ltd, China) are
   as follows: the wavelength of light 405 nm, spot size 50 µm, exposure
   time 6 s, exposure power 30 mW/cm^2, and layer thickness 25 µm. After
   the scaffold was cleaned with single distilled water, it was sterilized
   within an autoclave for 30 min at 121°C and then dried at 37°C for
   12 h. We measured the long axis of the elliptic hole on the side of the
   support as the pore diameter (40% 0.33 mm; 50% 0.45 mm; 60% 0.60 mm;
   and 70% 0.87 mm).

Microscopic observation and mechanical properties test of the scaffold

   Digital photos of the TPMS scaffold were taken from two perspectives
   using a Nikon digital camera: front and top views. A scanning electron
   microscope (JSM-IT500, JEOL, Japan) was used to observe the surface
   morphology of the scaffold. Using a universal material testing machine
   (INSTRON 3365, INSTRON, USA), the β-TCP scaffold was tested by axial
   compression to rupture, and the testing speed was 1 mm/min. Three
   samples were tested in each group to obtain the average value of their
   mechanical strength.

In vitro degradation testing

   The scaffolds were divided into four groups based on porosity, with
   three replicate samples in each group.The scaffolds were immersed in
   Tris-HCl buffer (0.05 M; pH ~ 7.4) at 37°C. At predetermined time
   intervals, they were retrieved, rinsed with distilled water, and dried.
   Changes in mass was monitored throughout the degradation period.

Cell proliferation detection

   BMSCs (CP-M131, Pricella, China) were seeded on TPMS scaffolds with
   different porosities in 48-well plates at a density of 1 × 10^5/well,
   and cultured in a cell incubator (37°C, volume fraction of 5% CO[2]).
   Three time points were established (1, 2, and 3 days). At each time
   point, 50 μL of CCK-8 solution was added to each sample and incubated
   in a cell incubator for 3 h. The CCK-8 solution in 48-well plate was
   transferred to a 96-well plate, and the absorbance value of each well
   was detected using the enzyme-labeled instrument (IMark, BIO-RAD, USA)
   at the wavelength of 450 nm. Each group of samples was repeated three
   times.

Cell adhesion detection

   After the cells and scaffolds were cultured for 24 h, the cells were
   subjected to live-dead staining. A 1 mL of Calcein–AM/PI mixed staining
   solution was added to each well and incubated in the dark at 37°Cand 5%
   CO[2] for 30 min. After washing with PBS, the samples were visualized
   under the inverted fluorescence microscope. The average total
   fluorescence intensity of the single well of each group of scaffolds
   was calculated using ImageJ software. BMSCs were cultured with the
   scaffold for 24 h, then the culture medium was aspirated from the well
   plate, moistened with PBS three times, and 1 mL of mixed staining
   solution of DAPI/F-actin was added to each well, and incubated in a
   cell incubator (37°C, volume fraction of 5% CO[2]) for 30 min in the
   dark. After PBS washing, the scaffold was moved to an inverted
   fluorescence microscope for observation. The samples were then
   transferred to a laser confocal dish, and three images were captured
   for each group on the same plane by a laser confocal microscope (TCS
   SP8, Leica, Germany), with magnifications of ×50, ×400, and ×1000,
   respectively. Then, the average value of the total fluorescence
   intensity of a single hole in each group was calculated by ImageJ
   software.

Preparation of critical mandibular bone defect model of rabbits

   All animal surgical operations were carried out according to the scheme
   approved by the Living Animal Teaching and Research Committee of
   Guangdong Medical University, with the license number GDY2204012.
   Fifteen 6-month-old healthy male New Zealand white rabbits weighing
   3 ± 0.5 kg were selected. They were randomly divided into four groups:
   40% porosity, 50% porosity, 60% porosity, and 70% porosity (n = 3 in
   each group). After anesthesia, the skin of the right submandibular
   region was prepared routinely, sterilized, and laid with sterile
   sheets, and a 2–3 cm incision was made parallel to the lower edge of
   the rabbit’s mandible from the posterior direction. The epidermis and
   subcutaneous tissues were incised layer by layer, and muscles were
   bluntly separated to expose the bone surface. A circular bone drill
   with a diameter of 8 mm was used to create a defect in the middle of
   the mandibular body, and cold physiological saline was used to reduce
   the temperature, to make a cylindrical bone defect with a diameter of
   8 mm and a depth of 4 mm. Scaffold materials with different porosity
   were implanted into the defect according to predetermined groups, and
   the muscle membrane, fascia, and skin were stitched in layers. After
   the operation, the 30,000 unit penicillin was injected intramuscularly
   3 days, and iodophor was used to disinfect the wound to prevent
   postoperative infection.

Micro-CT analysis

   2 months post operation, the rabbits were euthanized via anesthesia.
   Mandibular samples containing implanted scaffolds were collected and
   scanned by micro-CT. Rabbit mandibular samples were fixed in 10%
   neutral formalin buffer for 24 h, and then scanned by X-ray micro-CT
   (Xradia Versa610, ZEISS, Germany). Working conditions: voltage 70 kV,
   current 114 μA, scan resolution 17.5 μm, and rotation angle of 0°. The
   3D projection image was reconstructed from a stack of 2D images by
   using CTvox software (Skyscan, Bruker Corporation, Germany).

Histological analysis

   The tissues containing implants were collected and fixed with
   paraformaldehyde. After embedding in paraffin, the samples were sliced
   by microtomes (RM2235, Leica, Germany) with a thickness of 4 μm. Then,
   sections were stained with Hematoxylin-Eosin (H&E) and Masson solutions
   to observe the tissue structure, and to evaluate the effects of
   scaffolds with different porosity on bone growth. Stained samples were
   imaged by using the biological microscope (Leica, DM500, Germany)

Immunohistochemical analysis

   The sections were dewaxed to water, and the antigen retrieval was
   conduction using the high-pressure steam method. The expression levels
   of alkaline phosphatase (ALP) and osteoprotegerin (OPG) in bone tissues
   were detected by SABC immunohistochemical method. Stained samples were
   imaged using the biological microscope (Leica, DM500, Germany)

Differentially expressed genes (DEGs) analysis

   Genes differential expression analysis was performed by DESeq2 software
   between two different groups (and by edgeR between two samples). The
   genes with the parameter of false discovery rate (FDR) <0.05 and
   absolute fold change ⩾2 were considered differentially expressed genes.
   Differentially expressed genes were then subjected to enrichment
   analysis of GO functions and KEGG pathways.

Principal component analysis

   Principal component analysis (PCA) was performed with princomp function
   of R ([74]http://www.r-project.org/) in this experience. PCA is a
   statistical procedure that converts hundreds of thousands of correlated
   variables (gene expression) into a set of values of linearly
   uncorrelated variables called principal components. PCA is largely used
   to reveal the structure/relationship of the samples/datas.

GO enrichment analysis

   Gene Ontology (GO) is an international standardized gene functional
   classification system which offers a dynamic-updated controlled
   vocabulary and a strictly defined concept to comprehensively describe
   properties of genes and their products in any organism. GO has three
   ontologies: molecular function, cellular component, and biological
   process. The basic unit of GO is GO-term. Each GO-term belongs to a
   type of ontology.

   GO enrichment analysis provides all GO terms that significantly
   enriched in DEGs comparing to the genome background. First, all
   Differentially Expressed Genes (DEGs) were aligned with Gene Ontology
   (GO) terms available in the Gene Ontology database
   ([75]http://www.geneontology.org/). Subsequently, the gene counts for
   each term were computed, and significantly enriched GO terms among the
   DEGs were determined using the hypergeometric test, comparing them
   against the genome background.

Pathway enrichment analysis (KEGG)

   Genes usually interact with each other to play roles in certain
   biological functions. Pathway-based analysis helps to further
   understand genes biological functions. KEGG is the major public
   pathway-related database. Pathway enrichment analysis identified
   significantly enriched metabolic pathways or signal transduction
   pathways in DEGs comparing with the whole genome background.

Gene set enrichment analysis (GSEA)

   We performed gene set enrichment analysis using software GSEA (v4.1.0)
   and MSigDB to identify whether a set of genes in specific GO terms,
   KEGG pathways, DO terms (for Homo sapiens), Reactome (for a few model
   animals) shows significant differences in two groups. Briefly, we input
   gene expression matrix and rank genes by Signal2Noise normalization
   method. Enrichment scores and p value was calculated in default
   parameters. GO terms, KEGG pathways (DO terms, Reactome) meeting this
   condition with |NES|>1, NOM p < 0.05, FDR q < 0.25 were considered to
   be different in two groups.

Alternative splicing analysis

   rMATS (version 4.1.1; [76]http://rnaseq-mats.sourceforge.net) was used
   to identify alternative splicing events and analyze differential
   alternative splicing events between samples. We identified AS events
   with a false discovery rate (FDR) <0.05 in a comparison as significant
   AS events.

Real-time quantitative polymerase chain reaction (RT-qPCR)

   After co-culturing rabbit bone marrow mesenchymal stem cells with TPMS
   scaffolds for 14 days, the culture medium was aspirated from the wells
   of the plate. The wells were washed three times with 1×PBS solution,
   followed by the addition of 1 mL Trizol solution to each well. The
   scaffolds were repeatedly pipetted to extract total RNA from the cells
   on the scaffold. The extracted RNA was subjected to cDNA synthesis
   using the PrimeScript Reverse Transcription kit. Subsequently, RT-qPCR
   experiments were conducted using SYBR Green PCR mix (TaKaRa) and the
   LightCycler 480 II system (Roche, USA). The reaction conditions were as
   follows: initial denaturation at 95°C for 5 s; PCR quantification
   analysis at 95°C for 15 s, followed by 40 cycles of 60°C for 1 min;
   dissolution curve analysis at 95°C for 15 s, 60°C for 1 min, and 95°C
   for 1 min; and cooling at 50°C for 30 s. The relative expression levels
   of VEGFA (Vascular Endothelial Growth Factor A), S100A4 (S100 Calcium
   Binding Protein A4), YAP1 (Yes1 Associated Transcriptional Regulator),
   CSF1R (Colony Stimulating Factor 1 Receptor), and HES1 (Hes Family BHLH
   Transcription Factor 1) were determined by reverse transcribing 1 µg of
   total RNA into cDNA, and their relative expression levels were analyzed
   using the 2^−△△Ct method for quantification analysis.

Statistical methods

   The quantitative data is expressed as the mean ± standard deviation
   (SD). Use one-way ANOVA or Student-t test to compare the data.
   *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. p < 0.05 is
   considered to be statistically significant.

Results and discussion

Morphology and mechanical properties of TPMS scaffolds

   The TPMS structure was adopted to mimic the unique structure of
   cancellous bone with the aims to develop a biomimetic bone graft
   substitute. [77]Figure 1(a) shows a macroscopic image of the TPMS bone
   scaffolds with a porosity of 40%, 50%, 60%, and 70%. From the images,
   it can be observed that the different porosity scaffolds mimic
   different sizes of bone trabeculae. The surface morphology and internal
   structure of TPMS scaffolds with different porosity were observed via
   scanning electron microscopy. It was found that the 3D-printed
   scaffolds with three-period minimal curved surfaces exhibited a curved
   porous structure with uniform pore size distribution ([78]Figure 1(b)).
   The structure of the scaffold has continuous interlayer transitions
   with interconnected pore network. The surface of the scaffold is
   compact, and the ceramic particles are clearly visible. Research has
   shown that the mechanical properties of bone scaffolds are affected by
   the pore structure, with a clear correlation between the scaffolds
   porosity and mechanical properties.^[79]29,[80]30 Cancellous bone
   blocks were used as homogeneous materials in compression experiments,
   and various biomechanical properties were obtained as a reference.
   O’Mahony et al obtained the elastic modulus of cancellous bone from the
   edentulous mandible in three directions: the highest elastic modulus
   was in the proximal and distal directions, with an average of
   907 ± 849 MPa; the second was in the buccal and lingual directions,
   with an average of 511 ± 565 MPa; and in the upper and lower
   directions, with an average of 114 ± 78 MPa.^ [81]31 van Eijden’s study
   showed that the compressive strength of the mandible was between 1 and
   20 MPa.^ [82]32 The compressive strength and elastic modulus of the
   scaffolds with different porosity were measured by universal mechanical
   testing machine. The results showed that the compressive strength of
   the scaffolds with 40%, 50%, 60%, and 70% porosity were 3.19, 3.70,
   3.93, and 4.13 MPa, respectively ([83]Fig.ure 1(c)), with the 60%
   (p < 0.05) and 70% (p < 0.01) groups exhibiting a significantly
   enhanced compressive strength when compared to the 40% group. The
   compressive strength values obtained were similar to that of the
   mandible in the buccal-lingual direction, and scaffold with high
   porosity usually have more pores and channels, which helps to disperse
   stress and prevent crack growth. This structure can increase the
   strength of the material.The elastic modulus of the scaffolds with 40%,
   50%, 60%, and 70% porosity were 367.94, 481.42, 475.45, and 523.62 MPa
   respectively ([84]Figure 1(d)), with the 50% (p < 0.05), 60%
   (p < 0.05), and 70% (p < 0.01) groups exhibiting a significantly
   increased elastic modulus compared to the 40% group. The compressive
   strength values obtained for these different scaffold designs were in a
   similar range as the mandible. The biodegradation test reveals that
   degradation rates remain similar across various porosities, but 70%
   group (8.84%) showed higher mass loss after 6 months, with other
   scaffolds ranging from 5.49%~7.16%. The bone scaffold we prepared
   contains 85% of the photocurable monomer β-tricalcium phosphate and 15%
   HA. β-TCP has good degradation properties, but the crystal structure of
   hydroxyapatite is stable, and the degradation rate is relatively slow.^
   [85]33 The degradation rate is also affected by its large volume and
   compressive strength. At present, low replacement rate bone scaffolds
   have received attention from researchers and clinical doctors.^ [86]34
   The experimental results showed that the compressive strength and
   elastic modulus of the scaffold were similar to the cancellous bone,
   and the structure, elasticity and strength of the scaffold were in
   accordance with the physiological characteristics of cancellous bone,
   which is beneficial to supporting the biomechanical functions of bone
   tissue.

Figure 1.

   [87]Figure 1.
   [88]Open in a new tab

   (a) Digital photos of TPMS bone scaffold with porosities of 40%, 50%,
   60%, and 70%, (b) observing the micro-morphology of scaffolds with
   different porosities under scanning electron microscope, (c) the
   compressive strength of the scaffolds with different porosities, (d)
   the elastic modulus of the scaffolds with different porosities, and (e)
   the biodegradability of scaffolds with different porosities.

   *p < 0.05. **p < 0.01.

In vitro biocompatibility analysis

   In addition to the biomechanical properties of bone graft substitutes,
   the TPMS scaffolds should also be biocompatibility,^[89]35,[90]36
   promote cell adhesion and proliferation to facilitate effective tissue
   healing. The adhesion and proliferation of BMSCs on different
   structures were studied. To validate the biocompatibility of 3D-printed
   TPMS scaffolds, BMSCs were seeded onto the scaffolds for CCK-8 cell
   proliferation experiments. The results showed that 70% porous scaffolds
   exhibited the highest rate of proliferation at three time points,
   followed by the 60%, 50%, and 40% scaffolds ([91]Figure 2(b)). This
   profile may be due to the larger pore size of the macroporous
   scaffolds, which are more favorable to cell substance exchange.^ [92]37
   On the other hand, low porosity scaffolds are not conducive to the
   internal growth of cells, resulting in a smaller area of cell growth
   than the scaffold with a larger pore size. Cell-seeded scaffolds from
   each group were then stained using live-dead staining to explore
   long-term cell growth. Fluorescent microscopy in conjunction with
   fluorescence intensity analysis ([93]Figure 2(a) and ([94]c)), showed
   that 70% porosity had the highest cell proliferation rate, followed by
   60%, 50%, and 40% groups, indicating that scaffolds with greater
   porosity had improved cell proliferation effects than scaffolds with
   lower porosity.^ [95]38 The result from the CCK-8 assay and live/dead
   staining clearly indicate that the 70% porosity scaffold facilitated
   cellular proliferation to a greater degree when compared to the other
   scaffold groups. Laser confocal microscopy was employed to further
   investigate the influence of different scaffold architectures on
   cellular morphology within the construct ([96]Figure 3(a)). The cells
   within the 70% scaffolds exhibited the typical fibroblast-like
   morphology in addition to possessing the highest density of cells when
   compared to the other groups. Together, these findings indicate that
   the TPMS scaffolds were suitable for the early adhesion and
   proliferation of osteoblasts in the scaffolds of each group^ [97]39 It
   has been proved that, compared with the wooden crib structure, the TPMS
   scaffolds have greater specific surface area and surface continuity,
   greatly facilitating cell adhesion and growth, and thus higher cell
   activity during culture.^ [98]40 In addition, in order to better
   observe the growth and morphology of the cells on the scaffolds, and to
   verify that the porous TPMS scaffolds have good
   biocompatibility,^[99]41,[100]42 the cells on each group of scaffolds
   examined under the scanning electron microscope. As shown in
   [101]Figure 3(b), the cells in each group displayed the typical long
   fusiform morphology, highlighted by the blue arrow. Moreover, the
   images showed that the DLP-based 3D printing technology used in this
   study resulted in the scaffolds presenting a rough surface at the
   microscopic level, which is likely conducive to the attachment of
   osteoblasts’ pseudopodia.

Figure 2.

   [102]Figure 2.
   [103]Open in a new tab

   (a) Live-Dead staining of TPMS bone scaffold with 40%, 50%, 60%, and
   70% porosities, (b) column analysis diagram of CCK-8 experiment, and
   (c) histogram of average fluorescence intensity.

   *p < 0.05. **p < 0.01. ****p < 0.0001.

Figure 3.

   [104]Figure 3.
   [105]Open in a new tab

   (a) Laser confocal images of TPMS bone scaffolds with porosity of 40%,
   50%, 60%, and 70% under a 40-fold microscope (DAPI shows the nucleus,
   F-actin is stained with Phalloidin) and (b) scanning electron
   microscope images of TPMS bone scaffold with porosity of 40%, 50%, 60%,
   and 70%.

   Blue arrows refer to mouse bone marrow mesenchymal stem cells.

TPMS scaffolds facilitate the repair of mandibular defects in New Zealand
white rabbits

   To explore the regenerative effects of the TPMS scaffolds on mandibular
   repair, we established a defect model in the mandible of New Zealand
   white rabbits. An overview of the surgical procedure performed is shown
   in [106]Figure 4(a). The experimental animals recovered well after the
   operation, with no post-operative complications. Two months after
   surgery, the mandibles were extracted and analyzed by Micro-CT
   scanning. From [107]Figure 4(b), it can be seen that the de novo
   low-density cancellous bone tissue in the 70% porosity scaffold has a
   clearer trabecular structure when compared to the scaffold designs
   exhibiting lower porosities. When compared to the 70% scaffold, the 40%
   group exhibited reduced de novo bone formation particularly in the
   center of the constructs. This is likely due to the low porosity
   scaffolds restricting nutrient transport, neovascularization, and
   cellular ingrowth, critical processes in effective tissue healing.
   Moreover, smaller pores favor hypoxic conditions and induced
   osteochondral formation before osteogenesis, while large pores, that
   are well-vascularized, lead to direct osteogenesis.^ [108]43 The
   enhanced bone formation observed within the 70% scaffolds is consistent
   with the research that bone scaffolds with high porosity and parts
   close to the host bone tissue are more conducive to bone repair.^
   [109]44 Micro-CT analysis of ROI area bone tissue volume (BV;
   [110]Figure 4(c)), the total volume of the ROI area (TV; [111]Figure
   4(d)), the ratio of bone tissue to tissue volume can reflect changes in
   bone mass (BV/TV; [112]Figure 4(e)), and the average thickness of bone
   trabeculae (Tb.Th) was conducted on samples ([113]Figure 4(f)). The
   experimental results clearly show that the average thickness of newly
   formed bone tissue and trabeculae in the 70% porosity scaffold group
   was significantly higher than that of other groups ([114]Figure
   4(c)–(f); p < 0.0001). In addition to micro-CT analysis, the mandibles
   implanted with the TPMS bone scaffolds were extracted and assessed via
   histological (H&E and Masson trichrome staining) and
   immunohistochemical analysis (ALP and OPG). Several studies have
   highlighted the importance of scaffold porosity in facilitating bone
   ingrowth, allowing more cells to invade and providing a larger surface
   area, which is believed to contribute to higher bone-induced protein
   adsorption.^[115]45,[116]46 Our findings showed that the groups
   containing the 70% porosity scaffold displayed the best growth of
   cancellous bone ([117]Figure 5). Immunohistochemical analysis showed
   that all four scaffolds groups exhibited positive staining for ALP, an
   early marker of bone formation, with the 70% scaffold groups displaying
   more concentrated ALP positive areas in the newly formed bone tissue
   around the scaffold. Moreover, the average fluorescence intensity of
   OPG was most pronounced in the groups containing the 70% scaffold when
   compared to the other test groups ([118]Figure 6). In addition to
   osteogenic induction, angiogenesis is a critical process involved in
   the effective healing of bone defects.^ [119]47 The structure and
   composition of bone substitutes must allow vascularization by
   presenting interconnected porosity and favorable biochemical support.
   The latter can accelerate bone remodeling by strengthening capillaries
   to promote the colonization and retention of osteoblasts and
   nutrients.^[120]48 For bone substitutes to develop angiogenesis, pores
   seem to be essential in their structures. On the one hand, pore size
   directly plays a role in bone ingrowth,^ [121]49 ensuring cell
   colonization, migration, and transportation. Our findings showed that
   the 70% group displayed increased quantity of blood vessels (red arrow)
   within the constructs when compared to the other scaffold groups
   ([122]Figure 5). Collectively, these findings indicate the implantation
   of the TPMS scaffolds promoted effective bone regeneration at the
   mandibular defect site, with the 70% porosity bone scaffold exhibiting
   the best tissue healing. In future experiments, we expect to further
   optimize the scaffold material with a 70% porosity. Through
   modification and loading of biomaterials with bioactive factors, it is
   expected to promote the growth of new bone and vascular tissues in the
   early stage. Taken together, the results in this study demonstrate the
   considerable potential additively manufactured TPMS scaffolds provide
   as a bone graft substitute for mandibular defect repair.

Figure 4.

   [123]Figure 4.
   [124]Open in a new tab

   (a) Flow chart of mandibular defect modeling and TPMS-loaded bone
   scaffold in New Zealand white rabbits, (b) Micro-CT images of TPMS bone
   scaffolds with porosities of 40%, 50%, 60%, and 70%, (c) BV: ROI area
   bone tissue volume, (d) TV: The total volume of the ROI area, and (e)
   BV/TV: The ratio of bone tissue to tissue volume can reflect changes in
   bone mass. (f) Tb.Th: Average thickness of bone trabeculae.

   **p < 0.01. ***p < 0.001. ****p < 0.0001.

Figure 5.

   [125]Figure 5.
   [126]Open in a new tab

   Histological and immunohistochemical analysis of bone regeneration
   induced by TPMS scaffolds. Note: The yellow asterisks indicate mature
   bone tissue, the red arrows indicate new blood vessels, “T” indicates
   scaffolds.

Figure 6.

   [127]Figure 6.
   [128]Open in a new tab

   OPG immunofluorescent staining of bone regeneration induced by TPMS
   scaffolds. Note: DAPI staining blue represents the nucleus, green
   represents the OPG protein.

TPMS scaffolds foster bone repair via activation of the Hippo pathway

   To explore the mechanism of how 70% porosity bone scaffold influences
   BMSCs behaviors, RNA sequence analysis of mesenchymal stem cells
   cultured in these scaffolds for 2 weeks was analyzed. Principal
   components analysis of the data revealed that the 70% TPMS scaffold
   group samples had more similar gene expression profiles with each other
   than with the control samples ([129]Figure 7(a)).

Figure 7.

   [130]Figure 7.
   [131]Open in a new tab

   (a) Principal components (PC) analysis on tissues from TPMS scaffolds
   and tissues from control group, (b and c) Heatmaps of osteoblast
   differentiation-related genes, Angiogenesis-related factors and Hippo
   signaling regulation pathways genes expression after implant the
   scaffold for 2 weeks (red: high expression; blue: low expression), (d
   and e) GSEA enrichment analysis, (f) differential alternative splicing
   events between TPMS and control samples, (g) GO enrichment analysis.
   (h) KEGG pathway analysis of DEGs, (i) differentially expressed
   transcription factors family, and (j) RT-qPCR analysis of five genes
   (VEGFA, S100A4, YAP1, CSF1R, and HES1) related osteoblast
   differentiation, angiogenesis and Hippo signaling regulation pathways.

   A number of genes expression was up-regulated in 70% group. Concretely,
   the osteoblast differentiation-related genes (e.g. REST, BMPR1A, and
   SMAD3) and angiogenesis-related factors (e.g. VEGFA, HES1, PDGFC, and
   PDGFB) were significantly upregulated in the 70% group, indicating
   improved bone formation ability of BMSCs ([132]Figure 7(b)). Moreover,
   the gene expression of the Hippo signaling regulation pathways factors
   (e.g. YAP1, TCF7L2, and CSF1R) was significantly upregulated in the 70%
   group compared to control group ([133]Figure 7(c)). Hippo signaling
   pathway is important for the regulation of tissue growth, tissue
   homeostasis and regenerative repair.^[134]50–[135]52 The elevated gene
   expression of YAP1, PRKCE, SMAD3, and CSF1R indicated an increased
   propensity of differentiation into osteoblast cells within the 70%
   group.^[136]53,[137]54 The RT-qPCR analysis of VEGFA, S100A4, YAP1,
   CSF1R, and HES1 also confirmed the higher expression of
   angiogenesis-related factors and Hippo signaling pathway-related genes
   in the 70% groups ([138]Figure 7(j)). GSEA analysis found that the 70%
   group could promote the regulation of cell migration and blood vessel
   development ([139]Figure 7(d) and ([140]e)). These results together
   revealed the important role of scaffold on BMSCs behaviors and bone
   damage repair.

   [141]Figure 7(f) shows the differentially expressed statistics of
   alternative splicing which mainly containing mutually exclusive exon
   (MXE) and skipped exon (SE). Gene ontology (GO) analysis showed that
   70% TPMS scaffold induced the up-regulation of genes associated with
   integrin binding and positive regulation of NK T cell differentiation
   when compared with the control samples ([142]Figure 7(g)). Kyoto
   Encyclopedia of Genes and Genomes (KEGG) analysis also revealed gene
   enrichment in Hippo signaling pathway ([143]Figure 7(h)). Differential
   expression of transcription factors is mainly concentrated in ZBTB and
   zf-C2H2 family ([144]Figure 7(i)). These results emphasized that
   different cytoskeletal recombination and mechanical stress transduction
   of BMSCs in scaffolds with different pore morphology.

Conclusion

   In conclusion, this study developed a series of additively manufactured
   bioceramic scaffolds based on hydroxyapatite, mimicking the trabecular
   structure of cancellous bone for the regeneration of mandibular defects
   in New Zealand white rabbits. The experimental results showed that all
   four scaffolds displayed biocompatibility and good osteogenic capacity.
   In particular, the scaffolds exhibiting a 70% porosity showed
   significantly enhanced Hippo signaling pathway and mechanical
   properties, promoting osteogenic differentiation, and angiogenic in
   vitro and in vivo.

Footnotes

   Author contributions: J.Z, H.Z, and J.W conceived the study; H.Z and
   J.W performed the experiments; Z.L and Y.Y collected and J.H analyzed
   the data; H.Z and M.C wrote the manuscript; J.Z. and Q.L secured the
   funding for the research; X.L and K.M revised the manuscript. S.W
   responsible for TPMS bone scaffold design. All authors approved the
   final version of the manuscript.

   Availability of data and materials: All data needed to evaluate the
   conclusions in the paper are present in the paper and/or the
   Supplementary Materials. Additional data related to this paper may be
   requested from the authors.

   The author(s) declared no potential conflicts of interest with respect
   to the research, authorship, and/or publication of this article.

   Funding: The author(s) disclosed receipt of the following financial
   support for the research, authorship, and/or publication of this
   article: The study was supported by Guangdong Basic and Applied Basic
   Research Foundation (2020B1515120001), Dongguan Science and Technology
   of Social Development Program (20231800940152), Guangdong Medical
   University Undergraduate Innovation Experiment Project (FYDS001,
   FYDS002, FYDS003), and Guangdong University Student Innovation Project
   (S20202170520, S202210571094, S202210571046).

   Ethical approval: All animal surgical protocol was approved by the
   Institutional Animal Care and Use Committee at Guangdong Medical
   University (GDY2204012).

   ORCID iD: Hong Zhu Inline graphic
   [145]https://orcid.org/0000-0002-2528-0753

References