Download 3D printing scaffold combining with stem cells for bone defect repair

Abstract
Tissue engineering, which is the use of a combination of cells, engineering and materials
methods, and suitable biochemical factors to improve or replace biological functions,is the
favored strategy for the treatment of bone defects.Several stem cells with different
characteristics have been investigated.The most commonly used and further study in bone repair
is bone marrow mesenchymal stem cells.Secondly is the embryonic stem cells.There are many
kinds of scaffold materials which can be used for bone repair,including metals, ceramics,
polymers and composite.They can be divided into anorganic materials and organic
materials. Current research and development of 3D printing scaffold，combining with stem cells，
to repair bone defect，including composite collagen /rhBMP-2 chitosan microsphere modified
porous HA scaffold with human bone marrow mesenchymal stem cells,PLGA/HA scaffold
combined with human bone marrow mesenchymal stem cells and 3D collagen scaffold(BDTM
Three-Dimensional Collagen Composite Scaffold, Cat No. 354613, BD Bio-sciences) combined
with adipose derived stem cells.
Cell types involved in bone repair
At present, there are many kinds of stem cells which can be used for bone repair after the injury
of bone tissue, such as embryonic stem cells (hESCs), mesenchymal stem cells (hMSCs),
multipotent stem cells etc. Of those, Mesenchymal stem cells (MSCs) are non-haematopoietic
stromal stem cells that have many sources, such as bone marrow, periosteum, vessel walls,
adipose, muscle, tendon, peripheral circulation, umbilical cord blood, skin and dental tissues.[1]
Mesenchymal stem cells obtained from different pathways can be divided into bone marrow
mesenchymal stem cells (BMSCs), human amnion mesenchymal stem cells (HAMSCs), placental
mesenchymal stem cells (PMSCs), synovium-derived mesenchymal stem cells (SMSCs), umbilical
blood mesenchymal stem cells, skeletal muscle stem cells and adipose-derived stem cells
(ADSCs).
Above of those MSCs, the most commonly used and further study in bone repair is bone marrow
mesenchymal stem cells. Induced pluripotent stem cells although has a large differentiation
potential and convenient source, but the process of osteogenic differentiation in vitro is
cumbersome and complex. So there are few studies about it in bone repair. Consequently，we
just introduce the methods of BMSCs and ESCs in this article.
（Application of BMSCs in bone repair）
BM MSCs are commonly used for inducing bone repair, as they have a strong osteogenic potential,
low heterogeneity and are easily obtained by culturing iliaccrest aspirates.[2]
Cellular response by platelets, inflammatory cells and macrophages penetrating into the injured
bone promotes the migration of mesenchymal stem cells that differentiate into osteoblasts and
chondrocytes.[3]
Numerous groups are experimenting with tissue-engineered bone grafts in which MSCs
expanded in vitro are seeded onto osteoconductive scaffolds, which are then implanted at the
defect site.[4] Before being used in a scaffold, there are a variety of ways to place the cells onto
scaffolds, they are either introduced by systemic infusion, or growth on a scaffold and applied
directly to the site of the lesion, or genetically modified before being used in a scaffold.[5]
1.Expanded MSCs introduced by systemic infusion.
It has been proved that MSCs can migrate to the bone marrow after peripheral injection and
remain there for an extended duration, which is feasible and well tolerated for human.
2.Application of MSCs grown on scaffolds
Scaffolds serve as carriers for cultured MSCs before implantation. Scaffolds need to mimic the
natural environment of the bone matrix and should be safe to be used in clinical practice.
3.Genetically modified MSCs.
Numerous studies have been conducted in which MSCs are genetically modified to express an
osteogenic gene, causing bone induction in vivo.[5]
（Application of ESCs in bone repair）
ESCs were able to recapitulate the mesenchymal developmental pathway and were able to repair
the bone defect semi-autonomously, which can be used in tissue-engineered scaffolds.[4, 6] The
advantage of embryonic stem cells for bone repair is proliferating indefinitely，but the
heterogeneity of ESCs is larger than other stem cells and its direction of differentiation is not
fixed. The study also demonstrated that both hESCs and hMSCs can be directed to acquire
osteoblastic phenotype in vivo solely by biomaterial-based cues. Furthermore, hMSCs underwent
biomineralized scaffold-directed in vivo osteogenesis faster than hESCs, but both cell types
acquired similar extent of osteogenic differentiation by 8 weeks post-implantation.[7]
Scaffolds for bone healing
Besides autogenous bone and allogeneic tissue, surgeons and scientists have been developing
Scaffold materials to treat defects of the skeletal system. Scaffold materials contain two types:
anorganic materials and organic materials.[8, 9]
Metals generally include stainless steel, titanium and in 2010 magnesium alloy was discovered[10].
Excellent biocompatible, mechanical strength, processability and inexpensive those four major
excellences appear in Metals using. Metals should be excellent materials but three deficiencies
would be visible in application: stiffness, little biodegradability and prevent the native tissue.
Ceramics generally can be divided into four types--hydroxyapatite (HAp), tricalcium
phosphate(TCP), biphasic calcium phosphates (BCP) and calcium phosphate (CaP).The superior
performances of stability, degradation rate, modifiability, biocompatibility and bioactivation of
ceramics make it being worth-considering. However, low strength and brittleness is clearly seen
on Ceramics' applying.[11-13]
Bioglasses could be another quality choice with two traits: significantly high mechanical strength
and have connected pores which can connect the native tissue from mechanical stimulation[14].
Polymers is usually taken into consideration when Organic materials are talked about for its
outstanding characters: excellent biocompatibleand, easy to shape and have good degradable.
Also, shortage of immunological reactions and foreign body reactions was founded on Polymers
using[15].
3D printing scaffold combining with
stem cells for bone defect repair
Current research and development of 3D printing scaffold，combining with stem cells， to repair
bone defect
1.Repair of bone defect with composite collagen /rhBMP-2 chitosan microsphere modified
porous HA scaffold with human bone marrow mesenchymal stem cells
HBMSCs are the most popular stem cell in the bone repair field,especially with HA scaffold.
Material and method:Chitosan microspheres are prepared by emulsion crosslinking
method,while the human bone marrow derived mesenchymal stem cells are isolated and
cultured in a leaching solution with rhBMP-2 chitosan microspheres. The cytotoxicity and cell
proliferation experiments prove the biological compatibility of rhBMP-2 in vitro.The osteogenic
induction of human bone marrow mesenchymal stem cells was demonstrated by the detection of
alkaline phosphatase activity in rhBMP-2. A three-dimensional model of HA:With collagen as the
medium,rhBMP-2 chitosan microsphere ,cross-linked by vanillin,adhere to the surface of the
porous hydroxyapatite scaffolds and improve osteogenic ability of the human bone marrow
mesenchymal stem cells on the scaffold.Perform the stent implantation on the quadriceps muscle
pouches of the healthy New Zealand white rabbits.Then histological observation, micro-CT, and
statistical analysis demonstrated the formation of bone tissue.
Conclusion:That the porous HA scaffold of collagen /rhBMP-2 modified chitosan microspheres
can enhance the differentiation of human bone marrow mesenchymal stem cells to differentiate
into osteoblasts can be use to repair bone defect.
2.Repair of cartilage defect with PLGA/HA scaffold combined with human bone marrow
mesenchymal stem cells
There is another scaffold,PLGA/HA,which is used to repair the bone defect,while the stem cell
is hBMSC.
Material and method:Take proper amount of PLGA into the dioxane and then mixed with HA
solution, which is used to be the material and 3D printer printing the scaffold.HMSC,cultured
with complete medium and digested by trypsin,is made to be cell suspension,which is dropped to
the 6-hole-plate in sterile 3D scaffold. Add the appropriate amount of complete medium into the
scaffold ,and put it to the incubator with the circumstances of 37 degrees C, 5%CO2. Two or three
days later, draw out the complete culture medium, and add the complete culture medium of the
cartilage for culture.Then perform the stent implantation in rabbits in vivo.
By means of collagen II and proteoglycan detection show that PLGA/HA scaffold can promote
hMSCs grow into chondrocytes. and rabbit implanted under test for normal tissue, indicating that
hMSC were seeded PLGA / ha scaffold materials in accordance with the provisions.
Detection of normal tissue implant test will show that, the PLGA/HA scaffold with hMSC
conforms to the provisions.[16]
Conclusion:The PLGA/HA scaffold printed by 3D printer combined with human bone marrow
mesenchymal stem cells can be used to repair rabbit cartilage.
3.Repair of bone defect with 3D collagen scaffold(BDTM Three-Dimensional Collagen Composite
Scaffold, Cat No. 354613, BD Bio-sciences) combined with adipose derived stem cells
Besides hBMSC,there is another cell that has the potential to differentiate into
osteoblasts ,ADSC.
Material and method:ADSCs，isolated from lipoaspirates，are carefully cultured, expanded and
processed in flow cytometry.Seeding and planting of ADSC in collagen scaffold with the top down
method,after that,perform the osteogenic differentiation.With alkaline phosphatase staining,
phosphate staining, histological examination, immunohistochemistry, timing and quantitative
RT-PCR analysis, statistical analysis and scaffold planting evaluation,it turns out to be that the
feasibility of ADSC three dimensional osteogenesis in bone transplantation.,while BMSC as the
gold standard. [17]
Conclusion:3D collagen scaffold (BDTM Three-Dimensional Collagen Composite Scaffold, Cat No.
354613, BD Bio-sciences) combined with ADSC can be use to repair bone defect.
4.The potential of bone repair with 3D PS-Ti combined with adipose-tissue derived mesenchymal
stromal/stem cells,AMSC
PS titanium has good high friction coefficient and surface roughness, and the strength of the
material is similar to that of the elastic modulus of bone,which suggests that it can be fit into the
field of bone repair.
Material and method:PS-Ti discs,comprised of alloyed titanium, aluminum,vanadium, and
trace elements (Ti6Al4V),is made into three-dimensional metal shape.Adipose tissue derived
mesenchymal stem cells are incubated in a 6 well polystyrene plate coated with poly
methacrylate PS-Ti.After RNA extraction and RT-qPCR scanning electron microscope slides and
staining methods osteoblast-like cells will be observed.[18]
Conclusion:Adhesion between adipose mesenchymal stem cell PS Ti bone differentiation
successfully shows that bone cells could expand on metal，while maintaining the favorable
physical and chemical characteristics of metal Ti ,which improve the potential of implant
materials currently used in bone deficiency, diabetes, osteoporosis and other bone diseases.
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