뼈 조직 재생의 그래핀 계열 재료:전망 및 과제

Graphene Family Materials는 세포를 골형성 분화로 매개하고 생체 내에서 뼈 재생을 촉진합니다.

많은 학자들은 그래핀이 세포(예:치수 줄기 세포[64, 65], 골수 줄기 세포[8, 20, 66, 67], 치주 인대 줄기 세포[68])의 부착 및 증식을 허용할 뿐만 아니라 ], 인간 조골세포[69], 섬유아세포 세포[70], 종양 세포[43]) 명백한 세포독성의 징후가 없지만 초기 세포 조골 분화를 유도하고 높은 정도의 광물화를 생성할 수 있습니다[20, 64,65,66,67, 68]. 현재 수많은 팀에서 그래핀 계열 나노물질을 지지체 또는 지지체에 첨가제로, 기질 물질 표면에 코팅으로, 가이드 뼈 재생막 및 약물 전달 수단으로 적용하는 새로운 전략을 설계하기 위해 많은 연구를 했습니다. (그림 2). 그들은 그래핀 계열 물질을 사용하여 기질 물질의 특정 특성을 더욱 개선하고 기질 기반 복합 재료에 생리 활성 특성을 부여하려고 했습니다.

A. 뼈 재생을 위한 지지체 또는 지지체의 보강재로서의 그래핀 계열 재료. B. 골 재생을 위해 기판에 전사된 코팅으로서의 그래핀 계열 물질. C. 유도 골막의 첨가제로서의 그래핀 계열. D. 뼈 재생을 촉진하는 약물 전달 시스템으로서의 그래핀 계열 물질

비계 또는 비계의 보강재로서의 그래핀 패밀리 재료

뼈 조직 공학을 위한 가장 일반적인 전략은 뼈 재형성 및 재생의 자연적 과정을 시뮬레이션하는 것입니다. 이 전략은 3차원(3D) 생체 적합성, 생분해성, 골전도성 또는 골유도성 지지체에 의해 충족될 수 있습니다[3]. 이러한 종류의 스캐폴드는 골형성 세포 부착, 이동, 증식 및 분화뿐만 아니라 성장 인자의 운반체를 위한 세포외 기질(ECM)을 모방하는 이상적인 미세 환경을 제공할 수 있습니다[6]. 유망한 생체 적합성 지지체로서 그래핀은 세포 확산 및 기질에서 골 형성 분화에 사용할 수 있는 큰 표면적을 만들 수 있습니다[20]. 예를 들어, 인간 중간엽 줄기 세포(hMSC)의 배양 기질로 사용된 3D 그래핀 폼은 줄기 세포 생존력을 유지하고 골형성 분화를 촉진할 수 있다는 증거를 제공했습니다[66]. 또한, 3D 그래핀(3DGp) 지지체와 2D 그래핀(2DGp) 코팅은 더 높은 수준의 광물화 및 상향 조절된 뼈 관련 유전자 및 화학 인덕터를 사용하거나 사용하지 않고 그래핀의 단백질 [68].

요즘은 발판 역할을 하는 다양한 생체재료들이 버섯처럼 솟아나고 있다. 뼈 재생에 사용하기에 잠재적으로 적합한 합성 지지체에는 수산화인회석(HA)[71]과 같은 인산칼슘; β-인산삼칼슘(β-TCP)[72]; 폴리락트산(PLA)[73], 폴리글리콜산(PLGA)[74], 폴리카프로락톤(PCL)[75], 키토산(CS)[1], 콜라겐[76]과 같은 합성 또는 바이오 폴리머 ]; 및 위에서 언급한 재료의 합성물 [77, 78]. 그러나 이제 가장 중요한 관심사 중 하나는 비계의 기계적 특성입니다. 천연 뼈는 7-27GPa 범위의 영률 값을 갖는 초탄성 생체 역학적 특성을 나타내기 때문에 이상적인 지지체는 자연 뼈의 강도, 강성 및 기계적 거동을 모방해야 합니다. 그래핀 계열 재료는 기계적 특성을 강화하고 물리화학적 특성화를 개선하기 위해 스캐폴드에 강화 재료로 추가할 수 있습니다. 예를 들어 순수 PCL 스캐폴드의 인장 강도는 1.61MPa, 연신율은 122%, 영률은 7.01MPa입니다. GO(2%)를 추가하면 인장 강도가 3.50MPa로, 연신율이 131%로, 영률이 15.15MPa로 크게 증가했습니다[80].

그래핀 계열 재료를 강화 재료로 사용한 성공에 자극을 받아 많은 팀이 합성 또는 생체 고분자가 제공하는 생체 적합성과 그래핀 계열 재료의 놀라운 물리적 특성을 결합했습니다. 그들은 새로운 뼈 형성을 지원하고 유도하기 위해 개선된 기계적 특성, 적절한 다공성, 구조적 디자인 및 우수한 생체 적합성을 갖춘 이상적인 복합 지지체를 얻을 것으로 기대했습니다.

인산칼슘 기반 재료를 사용한 그래핀 제품군

인간의 뼈는 30%의 유기물(대부분 콜라겐)과 70%의 무기물(대부분 하이드록시아파타이트(HA; Ca₁₀) (PO₄ )₆ (OH)₂ ) [81, 82]. HA, β-TCP(β-tricalcium phosphate), CPC(calcium phosphate cements)와 같은 합성 인산칼슘계 재료는 뼈의 천연 광물상과 유사한 조성 및 구조와 우수한 골 형성성으로 인해 널리 사용되는 비계 재료입니다. 능력 [83,84,85]. 특히, HA의 우수한 골전도 및 골유도 능력 때문에[86], 골 결손 부위를 수복하기 위한 정형외과 또는 악안면 수술에서 인공 골 이식재로 오랫동안 널리 사용되어 왔다[11, 71]. 그러나 성형 어려움, 독특한 취성 및 낮은 파괴 인성과 같은 HA 재료의 고유한 단점을 개선해야 합니다[87, 88]. 그래핀 계열 재료 강화 HA 복합 재료가 개발되어 파괴 인성과 생물학적 성능이 크게 향상되었다고 보고되었습니다. 예를 들어, HA/그래핀 합성물은 HA 허용 강도를 부여하는 스파크 플라즈마 소결(SPS)에 의해 준비되었습니다[89]. Raucci et al. In situ sol-gel approach와 biomimetic approach의 두 가지 접근 방식으로 HA와 GO를 결합했습니다. in situ sol-gel 접근법으로 얻은 HA-GO는 골 형성 인자를 사용하지 않고 hMSC의 세포 생존 능력을 향상시키고 조골 세포 분화를 유도했습니다. 생체모방 접근법을 통해 형성된 HA-GO는 세포 생존과 증식을 지속시켰다[90]. 또한 환원그래핀옥사이드(rGO)는 HA의 보강재로도 사용될 수 있다. HA-rGO 복합 재료의 파괴 ​​인성은 3.94MPa m ^1/2 에 도달했습니다. , 순수 HA에 비해 203% 증가. HA-rGO는 인간 조골 세포의 알칼리성 인산 가수분해 효소(alkaline phosphatase, ALP) 활성에 의해 평가된 세포 증식 및 조골 분화를 향상시켰다[91]. 또한 Nie et al. 자가 조립을 통해 rGO 및 나노 하이드록시아파타이트(nHA) 3D 다공성 복합 지지체(nHA@rGO)를 성공적으로 합성했습니다. 자가 조립 과정을 유도하기 위해 가열된 nHA 물 현탁액과 혼합된 GO 용액. 마지막으로 반응 생성물을 동결 건조하여 3차원 다공성 지지체를 얻었다. nHA@rGO 스캐폴드는 쥐 뼈 중간엽 줄기 세포(rBMSC)의 세포 증식, ALP 활성 및 골형성 유전자 발현을 상당히 촉진할 수 있습니다. 그리고 생체 내 실험에서 20% nHA가 포함된 rGO(nHA@rGO) 다공성 지지체가 토끼의 원형 두개골 결손 치유를 가속화할 수 있음이 밝혀졌습니다[92]. 또한, 이중 성분뿐만 아니라 삼성분도 우수한 세포 적합성 및 향상된 친수성 및 기계적 특성으로 우수한 성능을 보였다[93,94,95].

인산삼칼슘의 유사체인 인산삼칼슘은 뼈회[Ca₃라고도 알려진 3차 인산칼슘입니다. (PO₄ )₂ ]. 그것은 쉽게 흡수될 수 있는 칼슘과 인의 풍부한 출처 역할을 합니다. Beta-tricalcium phosphate(β-TCP)는 생체 적합성이 높으며 결손 부위 내에 재흡수 가능한 연동 네트워크를 생성하여 치유를 촉진합니다[96]. Wu et al. 2D β-TCP-GO 디스크와 3D β-TCP-GO 스캐폴드를 성공적으로 합성했습니다. β-TCP 및 블랭크 대조군과 비교하여, 2D β-TCP-GO 디스크는 Wnt 관련 신호 전달 경로를 활성화하여 hBMSC의 증식, ALP 활성 및 골형성 유전자 발현을 유의하게 향상시켰으며, 이는 GO-의 우수한 시험관내 골자극 특성을 나타냅니다. 변형된 β-TCP[85]. Wnt 표준 신호 전달 경로는 세포 증식, 분화 및 형태 형성과 같은 세포 활동을 조절하는 데 중요한 역할을 하는 것으로 알려져 있습니다[97, 98]. 생체 내 연구는 3D β-TCP-GO 스캐폴드가 순수한 TCP 스캐폴드보다 두개골 결손에서 더 큰 새로운 뼈 형성을 갖는 것으로 나타났습니다(그림 3)[85]. GO-Cu 나노복합체 지지체(CPC/GO-Cu)가 통합된 새로운 스캐폴드, 인산칼슘 시멘트는 rBMSC의 접착 및 골형성 분화를 촉진했으며, 이는 Erk1/2를 활성화하여 rBMSC에서 Hif-1α의 발현을 상향조절할 수 있음을 확인했습니다. 신호전달 경로와 혈관내피성장인자(VEGF)와 BMP-2 단백질의 분비를 유도한다. 또한, CPC/GO-Cu 스캐폴드를 임계 크기의 두개골 결손이 있는 쥐에 이식한 결과, 스캐폴드(CPC/GO-Cu)가 결손 부위의 혈관신생 및 골형성을 유의하게 촉진하는 것으로 나타났습니다[99].

β-TCP 및 β-TCP-GO 스캐폴드에 대한 계획 설명은 생체 내 골형성을 자극했습니다. 8주 동안 토끼의 두개골 결함에 이식한 후 β-TCP 및 β-TCP-GO 지지체에 대한 생체 내 골 형성 능력의 마이크로 CT 분석 및 조직학적 분석. ref에서 재생산. [85] Journal of Carbon의 허가를 받아

키토산을 포함하는 그래핀 계열

Chitosan (CS), a highly versatile biopolymer, derived from the shells of crustaceans [1, 87], has a hydrophilic surface that promotes cell adhesion and proliferation and its degradation products are nontoxic. Chitosan is biocompatible, osteoconductive, hemostatic, and can be easily converted into the desired shapes [2]. Besides, chitosan can promote bone matrix of mineralization [1] and minimize the inflammatory response after implantation [100]. All properties above make chitosan especially attractive as a bone scaffold material. But the most challenging part is the obtainment of CS-based scaffolds with good mechanical properties and processability [101]. Interestingly, CS/GO scaffolds have high water-retention ability, porosity, and hydrophilic nature [101, 102]. The CS-based 3D materials were enriched with GO in different proportions (0.5 wt% and 3 wt%). The new developed CS/GO 3 wt% scaffold was expected to be ideally designed for bone tissue engineering applications in terms of biocompatibility and properties to promote cell growth and proliferation [103]. Another CHT/GO scaffold with 0, 0.5, and 3 wt.% GO were prepared by freeze-drying method. Similarly, the CS/GO 3 wt% scaffolds significantly enhanced the ALP activity in vitro and the new bone formation in vivo, suggesting a positive contribution of 3 wt% GO to the efficiency of osteogenic differentiation process (Fig. 4) [3]. All results proved that CS/GO scaffolds could be a feasible tool for the regeneration of bone defects, and the addition of a 3 wt% of GO to material composition could have a better impact on cell osteogenic differentiation.

아 ALP activity in mice calvaria defects implanted with CHT/GO and b histomophometric analysis of Masson Goldner trichrome-stained sections. ###p < 0.001 vs CHT; **p < 0.01 vs control; ***p  < 0.001 vs control. Reproduced from ref. [3] with permission from the Journal of Scientific Reports

Moreover, some tricomponent composites, such as CS, GO, and HA can release more Ca and P ions compared to the pure HA nanoparticles, displaying a high bioactivity of the composite scaffold [87]. Ravichandran et al. fabricated a unique composite scaffold, GO–CS–HA scaffold, and the incorporation of GO enhanced the tensile strength of CS up to 8.2 MPa and CS–HA to 10 MPa. And the results demonstrated that GO–CS–HA scaffolds facilitated cell adhesion and proliferation, meanwhile showed improved osteogenesis in in vitro tests [2]. Another tricomponent composite scaffold, containing CS, gelatin (Gn), and different concentrates of graphene oxide (0.1%, 0.25%, 0.5%, and 1% (w /v ) GO) showed better physic-chemical properties than CS/Gn scaffolds. The addition of GO at the concentration of 0.25% to CS/Gn scaffolds exhibited enhanced absorption of proteins, extensive apatite deposition. The 0.25% GO/CS/Gn scaffolds were cyto-friendly to rat osteoprogenitor cells, and they enhanced differentiation of mouse mesenchymal stem cells into osteoblasts in vitro (Fig. 5). The tibial bone defect filled with 0.25% GO/CS/Gn scaffolds showed the growth of new bone and bridging the defect area, indicating their biocompatible and osteogenic nature [104]. Thus, no matter bicomponent or tricomponent composites scaffolds, the addition of graphene family materials to chitosan can favorably improve the mechanical properties and regulate the biological response of osteoblasts, promoting osteogenic differentiation.

아 MTT assay after incubation of CS/Gn scaffolds and 0.25% GO/CS/Gn scaffolds with media for 48 h. The asterisk indicates a significant increase versus control, and the pound sign indicates a significant decrease versus control (p <0.05). ㄴ , ㄷ Expression of osteogenic-related genes (RUNX2, ALP, COL-1, and OC) in mMSCs cultured on CS/Gn scaffolds and 0.25% GO/CS/Gn scaffolds for 7 and 14 days measured by quantitative RT-PCR. Reproduced from ref. [104] with permission from the Journal of International Journal of Biological Macromolecules

Graphene Family with Other Synthetic or Bio-polymers

Sponge scaffolds of type I collagen, the major organic component of bone [81], have been clinically applied as scaffolds to regenerate bone tissue [105, 106]. Because collagen scaffolds (elastic moduli:14.6 ± 2.8 kPa) are relatively soft, the combination with GO is expected to enhance the elastic modulus of collagen scaffolds and to improve the osteogenic differentiation of MSCs for bone regeneration. The covalent conjugation of GO flakes to 3D collagen scaffolds (elastic moduli:38.7 ± 2.8 kPa) increased the scaffold stiffness by threefold and did not negatively affect the viability of BMSCs. The enhanced osteogenic differentiation observed on the stiffer scaffolds were likely mediated by BMSCs mechanosensing because the molecules involved in cell adhesion to stiff substrates were either upregulated or activated [107]. Moreover, the development of new biomaterials utilizing graphene family materials with high osteogenic capacity is urgently pursued (Table 2).

Up to now, these improved tricomponent systems for bone tissue engineering scaffolds possess good biocompatibility, which can promote cell attachment, proliferation, and have been reported mechanical properties matchable to those of natural bone. But the response to specific biological signals expressing, as well as the capabilities of enhancing cell differentiation and finally bone tissue regeneration, still needs to be explored further. Moreover, it has been reported that the pore structure (pore size, pore morphology, and pore orientation) and the elasticity of scaffolds were manipulated to regulate osteogenesis [108,109,110]. However, due to the complicated structure of porous and different elasticity accurately controlled of the scaffolds, it remains a major challenge to individually design specific pore architectures and elasticity 3D porous scaffolds that can stimulate bone regeneration. With the rapidly development of the science and technology, the emerging of the 3D-printing method may overcome this problem and open an avenue for bone tissue regeneration [85]. The in vitro bioactivity and excellent in vivo bone-forming ability of graphene family nanomaterials present a new prospect of developing a broad new type of multifunctional scaffolds for biomedical applications. Thus, we believe that the unraveled the molecular mechanisms behind will be revealed soon and graphene family materials still have attractive potential of applications in bone regeneration waiting us to explore.

Graphene Family Materials as Coating

Graphene family materials have been widely applied in diverse forms of medical applications for bone regeneration. As a coating, graphene family materials can be transferred on two dimensional (2D) flat non-metal or metal substrates to induce spontaneous osteogenic differentiation of several types of mesenchymal stem cells (MSCs) [64]. Nayak et al. transferred graphene to four 2D non-metal substrates (polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), glass slide, and silicon wafer with 300 nm SiO₂ (Si/SiO₂ ).) and investigated the influence of graphene on BMSCs differentiation. They summarized that the graphene coating was cytocompatible and contributed to enhance the osteogenic differentiation of BMSCs at a rate comparable to differentiation under the influence of BMP-2 in the osteogenic medium [20]. Similarly, Elkhenany et al. found that goat BMSCs, seeded on 2D graphene-coated plates underwent osteoblastic differentiation in culture medium without the addition of any specific growth factors [8]. Simultaneously, Lee et al. tried to explain the origin of how graphene coating could accelerate stem cell renewal and differentiation. They deemed that the strong noncovalent binding abilities of graphene allowed it to serve as a preconcentration platform for osteoblastic inducers, which facilitated BMSCs osteogenic differentiation [67]. The capability of graphene in modulating osteogenic differentiation is evident. How about its derivatives? GO coatings and rGO coatings all showed favorable cytocompatibility and enhanced spontaneous osteogenic differentiation by upregulating levels of ALP activity [111, 112].

Since titanium (Ti) and medical-grade Ti alloy have been extendedly applied in the orthopedic and dental fields [113,114,115], satisfactory osseointegration for titanium and its alloys is still a major challenge and need to be explored deeply in order to help the clinicians to promote the success or survive rate of implants and diminish the likely complications encountered after their placement [114, 116, 117]. Graphene family materials coated titanium and its alloys, serving as a new method to improve their capabilities of osseointegration at the tissue-implant interface, attracted widespread attention. For example, GO-coated titanium enhanced cell proliferation, upregulated levels of ALP activity and gene expression level of osteogenesis-related markers, and promoted the protein expression of BSP, Runx2, and OCN [117]. Qiu et al. made different thickness GO coatings on the pure titanium surfaces respectively by cathodal electrophoretic deposition. Interestingly, with the increasing thickness of GO, the ALP-positive areas improved, ECM mineralization increased [118]. Moreover, Zeng et al. firstly fabricated GO/HA composite coatings by electrochemical deposition technique on Ti substrate. The addition of GO facilitated both the crystallinity of deposited apatite particles and the bonding strength of the as-synthesized composite coatings [119]. It is well known that hydrophilic surface is biocompatible compared to hydrophobic surface. In the case of rGO coating, the rapid adsorption of serum protein improves hydrophilia of graphene surface and enhances cell adhesion. Jia et al. used evaporation-assisted electrostatic assembly and one-pot assembly to fabricate 2D GO-coated Ti and rGO-coated Ti, with tailored sheet size and surface properties. Compared to the contact angle of titanium (60.4°), the contact angle of GO-coated Ti and rGO-coated Ti were 20° and 14.2°, respectively, indicating the successful interfacial assembly of graphene and excellent wettability properties. The rGO-coated Ti elicited better cell adhesion and growth than bulk GO, while the latter evoked higher activity of osteogenic differentiation [120].

Osseointegration is a complicated biological process determined by the surface properties of implants [114]. The graphene-based coatings above all lack 3D morphology. The 3D porous surface structure of coating can mimic the special macrostructures of the nature bone tissues [115]. Qiu et al. first synthesized 3D porous graphene-based coating on the pure titanium plates (GO@Ti and rGO@Ti). Water contact angles showed super hydrophilic surfaces of GO@Ti and rGO@Ti. Surface wettability exerts great effect on the biocompatibility of materials, which is strongly related to biomolecules adsorption [121]. GO@Ti and rGO@Ti both showed the excellent cytocompability and the optimal capability of osteoinduction [39]. Morin and his co-workers even transferred single or double chemical vapor deposition (CVD) grown graphene coatings onto 3D objects with differences in 3D geometries and surface roughness, such us dental implant, locking compression plate and mandible plate (Fig. 6) [64]. CVD is a very stable coating fabrication method, with substrate-independent properties and versatile surface functionalization. Besides, surface active CVD coatings are good platforms for immobilizing biomolecules, which is very important to bone regeneration [122].

아 The calvarial defects of rats were enclosed with a GO-Ti membrane. ㄴ New bone formation of the rat calvarial defects after the implantation of Ti or GO-Ti membrane at postoperative week 8. *p  < 0.05 vs control; #p < 0.05 vs Ti. ㄷ Images of HE staining of the rat calvarial defects after the implantation of Ti or GO-Ti membrane at postoperative week 8. Reproduced from ref. [128] with permission from the Journal of Applied Spectroscopy Reviews

Overall, the strategy of applying graphene family materials as coating onto a surface is charming. Through currently available techniques or methods, such as CVD [123], electrochemical deposition [119], with diverse substrates (e.g. polymers, metals), graphene, and its derivatives can be obtained efficiently, with dimensions ranging from nanometer to macroscopic scales [120]. Then, graphene family nanomaterials can be transferred onto the substrate, either as 2D coatings/films/sheets or 3D porous structures of coating, to enable the binding of biomolecules, absorb the serum protein, and facilitate osteogenic differentiation of stem cells. But the different physical and chemical properties of the substrates and the type or frequent use of chemical inducers for osteogenic differentiation (e.g., dexamethasone, bone morphogenetic protein-2) that may cover up the effects exerted by graphene family materials alone [65]. Therefore, these methods still require to be well-directly improved and further studied.

Graphene Family as an Additive in Guided Bone Membrane

Barrier membranes are standardly used in oral surgical procedures, applying in guided tissue regeneration (GTR) and guided bone regeneration (GBR), for the treatment of periodontal bone defects and peri-implant defects, as well as for bone augmentation [124, 125]. GBR is considered to be one of the most promising methods for bone tissue regeneration. The concept of GBR is using a non-resorbable or absorbable membrane serving as a barrier to prevent the ingrowth of soft connective tissue into the bone defect and offer a space to “guide” the bone reconstruction [126, 127]. An ideal GBR membrane should have excellent biocompatibility and mechanical property to promote the regeneration of bone tissues and prevent soft-tissue ingrowth. Ti membrane is a non-resorbable membrane with excellent mechanical properties for the stabilization of bone grafts. Park et al. fabricated GO-coated Ti (GO-Ti) membranes, with increased roughness and higher hydrophilicity. GO endowed the pure Ti membranes better biocompatibility and enhanced the attachment, proliferation, and osteogenesis of MC3T3-E1 in vitro. Moreover, GO-Ti membranes were implanted into rat calvarial defects (Fig. 6) and new bone formation significantly in full-thickness calvarial defects without inflammatory responses was observed [128].

However, non-resorbable membranes need to be removed by a second operation. Thus, a resorbable membrane is recommended owing to avoid a second intervention during operation, which can diminish the risk of infection and the loss of the regenerated bone. But the resorbable membranes made of collagen or chitosan usually has poor mechanical property. The addition of graphene family materials improves the weaknesses of resorbable membrane. For instance, De et al. attempted to prepare absorbable collagen membranes enriched with different concentrations of GO. The presence of GO on the membrane altered the mechanical features of the membrane, by conferring lower deformability, improving stiffness, and increasing roughness [129]. Tian et al. made 3D rGO (3D-rGO) porous films, which can accelerate cell viability and proliferation, as well as significantly enhanced ALP activity and osteogenic-related gene expressions [130].

Although pristine graphene is basically incompatible with organic polymer to form homogeneous composite, and even decrease the cell viability in some cases if the amount of graphene is excessive [131]. The incorporation of graphene family materials can enhance the bioactivity and mechanical properties of composite membranes. Because of the potent effects on altering mechanical drawbacks, stimulating osteogenic differentiation, and exhibiting superior bioactivity, graphene family material-modified membranes can be applied effectively to GBR.

Graphene Family Materials as Drug Delivery System (DDS)

Due to their small size, intrinsic optical properties, large specific surface area, low cost, and useful noncovalent interactions with aromatic drug molecules, graphene family materials exhibit excellent efficacy as delivery vehicles of genes and biomolecules. Moreover, simple physisorption via π-π stacking, hydrogen bonding, and electrostatic interaction is able to assist in high drug loading of hydrophobic drugs without compromising potency or efficiency [38]. The therapeutic efficacy of drugs is always related to the drug delivery carrier, which should enable the loading of large doses, controlled release, and retention of the bioactivity of the therapeutic proteins [132]. At present, anticancer drugs, including doxorubicin [133,134,135,136,137], paclitaxel [138, 139], cisplatin [140], and methotrexate [141, 142] loaded by graphene family nanomaterials showed amazing cancerous effect for the selective killing of cancer cells.

For better bone regeneration, we sometimes need the help of osteogenic drug or macromolecular osteogenic protein. It was reported that the adsorbed drugs or loaded growth factors on graphene or its derivatives could enhance the osteogenic differentiation of cells due to the increased local concentration [143]. For example, simvastatin (SIM) chosen as a model drug was loaded on the 3D porous scaffolds, which were made of silk fibroin (SF) and GO. SIM is an inhibitor of the competitive 3-hydroxy-3-methyl coenzyme A (HMG-CoA) reductase [144]. The effects of SIM on bone formation are associated with an increase in the expression of bone morphogenetic protein-2 (BMP-2) mRNA and enhanced the vascular endothelial growth factor (VEGF) expression [145, 146]. SIM can release sustainedly (30 days), and the release rate was relevant to the GO content within the scaffolds. In vitro, compared with the blank scaffolds, the SF/GO/SIM showed better biocompatibility, and the cells cultured on them exhibited faster proliferation rate [147]. Dexamethasone (DEX) is an osteogenic drug for which can facilitate osseointegration. Jung et al. firstly loaded DEX on rGO-coated Ti by π-π stacking. The loading efficiency of DEX on rGO-Ti was 31% after drug loading for 24 h and only 10% of total loaded DEX was released for 7 days, indicating that the drug delivery system can induce a long-term stimulation of stem cells for osteogenic differentiation. The DEX/rGO-Ti significantly facilitated MC3T3-E1 cells growth and differentiation into osteoblasts [143]. Similarly, Ren et al. also employed the GO-Ti and rGO-Ti as drug vehicles to absorb DEX. The presence of DEX-GO and DEX-rGO helped to promote the cell proliferation and largely enhanced osteogenic differentiation [115]. The graphene family materials coating on Ti alloys with controlled drug delivery can stimulate and enhance cellular response around implant surface to reduce the osseointegration time, expected to be applied for various dental and biomedical applications [143].

Not only small molecular osteogenic drug, but also macromolecular proteins can be loaded by graphene family materials for bone regeneration. Bone morphogenetic proteins (BMPs) are the most potent osteoinductive protein for bone regeneration. Thus, BMP-2 was loaded on the surface of Ti/GO through π-π stacking and the interaction between negatively charged carboxylic groups at the edges of GO and positively charged amino acid residues of BMP-2 [132]. Ti/GO/BMP-2 exhibited the high loading and the sustained release of BMP-2 with preservation of its 3D conformational stability and bioactivity. In vitro, the capability of Ti/GO/BMP-2 is to enhance osteogenic differentiation of hBMSCs. In a mouse calvarial defect model, compared to Ti/BMP-2 implants, Ti/GO/BMP-2 implants around had much more extensive bone formation [132]. Xie et al. used GO-modified hydroxyapatite (HA) and GO-modified tricalcium phosphate (TCP) as an anchor for adsorbing BMP-encapsulated BSA- nanoparticles (NPs) respectively. The charge balance and BMP-2 sustained release capability of the new scaffolds synergistically improved BMSCs proliferation, differentiation, and bone regeneration in vivo [148]. Poor osteointegration and infection are the most serious complications leading to failures of Ti implantation [10]. Han et al. incorporated GO onto polydopamine (PDA)-modified Ti scaffolds. Then, BMP-2 and vancomycin (Van) were separately encapsulated into gelatin microspheres (GelMS). After that, drug-containing GelMS were loaded on GO/Ti scaffolds and anchored by the functional groups of GO (Fig. 7). The new scaffolds were endowed with dual functions of inducing bone regeneration and preventing bacterial infection [149]. Substance P (SP) is a highly conserved 11 amino acid neuropeptide [150], involved in many processes, such as the regulation of inflammation, wound healing, and angiogenesis, and it is expected to promote MSC recruitment to the implants [151]. Therefore, apart from BMP-2, La et al. added this peptide, SP, on the surface of GO-coated Ti. The dual delivery system via GO-coated Ti showed sustained release of BMP-2 and SP and the potential of SP for inducing migration of MSCs. In vivo, Ti/GO/SP/BMP-2 group showed the greater new bone formation in the mouse calvaria than Ti/GO/BMP-2 group may be due to the MSCs recruitment by SP to the implants [152].

Schematics and scanning electron micrographs of the preparation the new GO/Ti scaffold:BMP2- and Van-loaded CGelMS were immobilized on the GO/Ti scaffold through electrostatic interactions between the functional groups of GO and CGelMS. Reproduced from ref. [149] with permission from the Journal of Biomaterials Science

Currently, more and more teams get down to designing new drug delivery system to improve the practical applications. The loading of large doses, controlled release, and retention of the bioactivity of the therapeutic proteins are still difficulty in research on drug delivery system.