Analysis of OPLA scaffolds for bone engineering constructs using human jaw periosteal cells

For bone regeneration constructs using human jaw periosteal cells (JPC) the extent of osteoinductive ability of different three-dimensional scaffolds is not yet established. We analyzed open-cell polylactic acid (OPLA) scaffolds for their suitability as bone engineering constructs using human JPC. Cell adhesion and spreading was visualized on the surface of scaffolds by scanning electron microscopy. JPC proliferation within OPLA scaffolds was compared with proliferation within collagen and calcium phosphate scaffolds. We found a significant increase of proliferation rates in OPLA scaffolds versus Coll/CaP scaffolds at three time points. Live-measurements of oxygen consumption within the cell-seeded scaffolds indicate that the in vitro culturing time should not exceed 12–15 days. OPLA scaffolds, which were turned out to be the most beneficial for JPC growth, were chosen for osteogenic differentiation experiments with or without BMP-2. Gene expression analyses demonstrated induction of several osteogenic genes (alkaline phosphatase, osterix, Runx-2 and insulin-like growth factor) within the 3D-scaffolds after 12 days of in vitro culturing. Element analysis by EDX spectrometry of arising nodules during osteogenesis demonstrated that JPC growing within OPLA scaffolds are able to form CaP particles. We conclude that OPLA scaffolds provide a promising environment for bone substitutes using human JPC.     

Electrospun poly(vinylidene fluoride-trifluoroethylene)/zinc oxide nanocomposite tissue engineering scaffolds with enhanced cell adhesion and blood vessel formation

Piezoelectric materials that generate electrical signals in response to mechanical strain can be used in tissue engineering to stimulate cell proliferation.Poly (vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)),a piezoelectric polymer,is widely used in biomaterial applications.We hypothesized that incorporation of zinc oxide (ZnO) nanoparticles into the P(VDF-TrFE) matrix could promote adhesion,migration,and proliferation of cells,as well as blood vessel formation (angiogenesis).In this study,we fabricated and comprehensively characterized a novel electrospun P(VDF-TrFE)/ZnO nanocomposite tissue engineering scaffold.We analyzed the morphological features of the polymeric matrix by scanning electron microscopy,and utilized Fourier transform infrared spectroscopy,X-ray diffraction,and differential scanning calorimetry to examine changes in the crystalline phases of the copolymer due to addition of the nanoparticles.We detected no or minimal adverse effects of the biomaterials with regard to blood compatibility in vitro,biocompatibility,and cytotoxicity,indicating that P(VDF-TrFE)/ZnO nanocomposite scaffolds are suitable for tissue engineering applications.Interestingly,human mesenchymal stem cells (hMSCs) and human umbilical vein endothelial cells cultured on the nanocomposite scaffolds exhibited higher cell viability,adhesion,and proliferation compared to cells cultured on tissue culture plates or neat P(VDF-TrFE) scaffolds.Nanocomposite scaffolds implanted into rats with or without hMSCs did not elicit immunological responses,as assessed by macroscopic analysis and histology.Importantly,nanocomposite scaffolds promoted angiogenesis,which was increased in scaffolds pre-seeded with hMSCs.Overall,our results highlight the potential of these novel P(VDF-TrFE)/ZnO nanocomposites for use in tissue engineering,due to their biocompatibility and ability to promote cell adhesion and angiogenesis.     

3D Jet Writing: Functional Microtissues Based on Tessellated Scaffold Architectures

The advent of adaptive manufacturing techniques supports the vision of cell‐instructive materials that mimic biological tissues. 3D jet writing, a modified electrospinning process reported herein, yields 3D structures with unprecedented precision and resolution offering customizable pore geometries and scalability to over tens of centimeters. These scaffolds support the 3D expansion and differentiation of human mesenchymal stem cells in vitro. Implantation of these constructs leads to the healing of critical bone defects in vivo without exogenous growth factors. When applied as a metastatic target site in mice, circulating cancer cells home in to the osteogenic environment simulated on 3D jet writing scaffolds, despite implantation in an anatomically abnormal site. Through 3D jet writing, the formation of tessellated microtissues is demonstrated, which serve as a versatile 3D cell culture platform in a range of biomedical applications including regenerative medicine, cancer biology, and stem cell biotechnology.     

Aligned Nanofiber Topographies Enhance the Differentiation of Adult Renal Stem Cells into Glomerular Podocytes

The use of adult stem/progenitor cells is a challenge in current research about kidney functional regeneration. In this framework, material‐induced stem cell differentiation can become a new paradigm to promote advances in therapies. Here, the effects of both isotropic and anisotropic fibrous topographies on the podocyte differentiation of adult renal stem cells (RSCs) from human donors are investigated. The proliferation rate of RSCs is analyzed, immunofluorescence and genetic analyses of specific podocyte markers (Wilms’ tumor 1 gene, nephrin, and podocin) are performed to assess the differentiation level on the scaffolds. The studied markers are over‐expressed in RSCs cultured on aligned fibers compared to cells cultured on either protein‐functionalized films or randomly oriented fibers. In addition, RSCs cultured on aligned fibers are found to differentiate toward podocyte precursors even in basal medium conditions, thus highlighting scaffold‐induced commitment without exogenous chemicals or cellular reprogramming. Aligned polymer fiber scaffolds which provide instructive cues for RSC differentiation might lead to new biomimetic systems for renal stem cell engineering.

     

Mussel Adhesion‐Inspired Reverse Transfection Platform Enhances Osteogenic Differentiation and Bone Formation of Human Adipose‐Derived Stem Cells

Using small interfering RNA (siRNA) to regulate gene expression is an emerging strategy for stem cell manipulation to improve stem cell therapy. However, conventional methods of siRNA delivery into stem cells based on solution‐mediated transfection are limited due to low transfection efficiency and insufficient duration of cell‐siRNA contact during lengthy culturing protocols. To overcome these limitations, a bio‐inspired polymer‐mediated reverse transfection system is developed consisting of implantable poly(lactic‐co‐glycolic acid) (PLGA) scaffolds functionalized with siRNA‐lipidoid nanoparticle (sLNP) complexes via polydopamine (pDA) coating. Immobilized sLNP complexes are stably maintained without any loss of siRNA on the pDA‐coated scaffolds for 2 weeks, likely due to the formation of strong covalent bonds between amine groups of sLNP and catechol group of pDA. siRNA reverse transfection with the pDA‐sLNP‐PLGA system does not exhibit cytotoxicity and induces efficient silencing of an osteogenesis inhibitor gene in human adipose‐derived stem cells (hADSCs), resulting in enhanced osteogenic differentiation of hADSCs. Finally, hADSCs osteogenically committed on the pDA‐sLNP‐PLGA scaffolds enhanced bone formation in a mouse model of critical‐sized bone defect. Therefore, the bio‐inspired reverse transfection system can provide an all‐in‐one platform for genetic modification, differentiation, and transplantation of stem cells, simultaneously enabling both stem cell manipulation and tissue engineering.     

Processing and characterization of porous alumina scaffolds

Bioceramic materials are used for the reconstruction or replacement of the damaged parts of the human body. In this study an improved procedure is described for producing ceramic scaffolds with controlled porosity. Bioinert alumina ceramic was used to make porous scaffolds by using indirect fused deposition modeling (FDM), a commercially available rapid prototyping (RP) technique. Porous alumina samples were coated with hydroxyapatite (HAp) to increase the biocompatibility of the scaffolds. Initial biological responses of the porous alumina scaffolds were assessed in vitro using rat pituitary tumor cells (PR1). Both porous alumina and HAp coated alumina ceramics provided favorable sites for cell attachments in a physiological solution at 37 °C, which suggests that these materials would promote good bonding while used as bone implants in vivo. Based on these preliminary studies, similar tests were performed with human osteosarcoma cells. Cell proliferation studies show that both the ceramic materials can potentially provide a non-toxic surface for bone bonding when implanted in vivo.     

3D culturing of human adipose-derived stem cells enhances their pluripotency and differentiation abilities

Human mesenchymal stem cells,such as human adipose-derived stem cells(hASCs),are typically cultured on a two-dimensional(2 D)monolayer material surface,on which 2 D culturing methods are easily performed and time-saving.However,hASCs usually suffer from decreased pluripotency and differentiation ability when cultured with a 2 D monolayer culturing method compared to hASCs cultured with a three-dimensional(3 D)culturing method,such as suspension culture.In this study,we evaluated whether the pluripotency and differentiation ability of hASCs can be reversibly changed during sequential cultivation with 2 D and 3 D culturing processes.The hASCs cultivated with a 3 D culturing process after 2 D culture showed at least 2-fold enhanced pluripotency(Sox2,Nanog,and OCT4)compared with that of hASCs cultured with the 2 D culture process alone.Furthermore,hASCs obtained from the 3 D culture process expressed increased levels of differentiation markers of chondrocytes and osteoblasts compared with hASCs obtained from the 2 D culture process when hASCs were induced to differentiate.However,their pluripotency and differentiation ability were extensively reduced when hASCs were shifted from 3 D culture to 2 D culture and vice versa,which indicates that hASCs show reversibility in terms of their pluripotency and differentiation ability depending on their environment in 2 D and 3 D culture.The reversibility of pluripotency and differentiation ability were found to last for at least 5 passages in culture during the alternative and sequential culture of cells with 2 D and 3 D culturing processes.Our study revealed the importance of the culture microenvironment in maintaining the pluripotency and differentiation ability of hASCs,which may reduce the effects of the aging process in hASCs.We discuss whether the environment of stem cell culture(i.e.,2 D or 3 D cultivation)can affect stem cell fate in terms of pluripotency and differentiation reversibility.     

Porous nanocellulose gels and foams: Breakthrough status in the development of scaffolds for tissue engineering

We report on the latest scientific advances related to the use of porous foams and gels prepared with cellulose nanofibrils (CNF) and nanocrystals (CNC) as well as bacterial nanocellulose (BNC) – collectively nanocelluloses – as biomedical materials for application in tissue regeneration. Interest in such applications stems from the lightweight and strong structures that can be efficiently produced from these nanocelluloses. Dried nanocellulose foams and gels, including xerogels, cryogels, and aerogels have been synthesized effortlessly using green, scalable, and cost-effective techniques. Methods to control structural features (e.g., porosity, morphology, and mechanical performance) and biological interactions (e.g., biocompatibility and biodegradability) are discussed in light of specific tissues of interest. The state-of-the-art in the field of nanocellulose-based scaffolds for tissue engineering is presented, covering physicochemical and biological properties relevant to these porous systems that promise groundbreaking advances. Specifically, these materials show excellent performance for in vitro cell culturing and in vivo implantation. We report on recent efforts related to BNC scaffolds used in animal and human implants, which furthermore support the viability of CNF- and CNC-based scaffolds in next-generation biomedical materials.     

CAD/CAM scaffolds for bone tissue engineering: investigation of biocompatibility of selective laser melted lightweight titanium

The objective of the current in‐vitro study was to evaluate the biocompatibility of a new type of CAD/CAM scaffold for bone tissue engineering by using human cells. Porous lightweight titanium scaffolds and Bio‐Oss® scaffolds as well as their eluates were used for incubation with human osteoblasts, fibroblasts and osteosarcoma cells. The cell viability was assessed by using fluorescein diazo‐acetate propidium iodide staining. Cell proliferation and metabolism was examined by using MTT‐, WST‐Test and BrdU‐ELISA tests. Scanning electron microscope was used for investigation of the cell adhesion behaviour. The number of devitalised cells in all treatment groups did not significantly deviate from the control group. According to MTT and WST results, the number of metabolically active cells was decreased by the eluates of both test groups with a more pronounced impact of the eluate from Bio‐Oss®. The proliferation of the cells was inhibited by the addition of the eluates. Both scaffolds showed a partial surface coverage after 1 week and an extensive to complete coverage after 3 weeks. The CAD/CAM titanium scaffolds showed favourable biocompatibility compared to Bio‐Oss® scaffolds in vitro. The opportunity of a defect‐specific design and rapid prototyping by selective laser melting are relevant advantages in the field of bone tissue engineering and regenerative medicine.Inspec keywords: calcium compounds, scanning electron microscopy, adhesion, titanium, CAD/CAM, tissue engineering, bone, biomedical materials, cellular biophysics, biomechanics, laser materials processing, meltingOther keywords: bone tissue engineering, human cells, porous lightweight titanium scaffolds, human osteoblasts, osteosarcoma cells, cell viability, fluorescein diazo‐acetate propidium iodide staining, cell proliferation, MTT tests, WST‐Test, BrdU‐ELISA tests, cell adhesion, devitalised cells, metabolically active cells, biocompatibility, selective laser melting, CAD‐CAM scaffolds, cell metabolism, scanning electron microscopy, Ti     

In vitro biocompatibility assessment of PHBV/Wollastonite composites

Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing bone tissue engineering scaffolds, the in vitro biocompatibility of degradable and bioactive composites consisting of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and wollastonite (W) was studied by culturing osteoblasts on the PHBV/W substrates, and the cell adhesion, morphology, proliferation, and alkaline phosphatase (ALP) activity were evaluated. The results showed that the incorporation of wollastonite benefited osteoblasts adhesion and the osteoblasts cultured on the PHBV/W composite substrates spread better as compared to those on the pure PHBV after culturing for 3 h. In the prolonged incubation time, the osteoblasts cultured on the PHBV/W composite substrates revealed a higher proliferation and differentiation rate than those on the pure PHBV substrates. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composites increased from 10 to 20 wt%. All of the results showed that the addition of wollastonite into PHBV could stimulate osteoblasts to proliferate and differentiate and the PHBV/W composites with wollastonite up to 20 wt% were more compatible than the pure PHBV materials for bone repair and bone tissue engineering.     

Gelatin sponges (Gelfoam ® ) as a scaffold for osteoblasts

Gelatine sponge because of its flexibility, biocompatibility, and biodegradability, has the potential to be used as a scaffold to support osteoblasts and to promote bone regeneration in defective areas. This study aimed to determine osteoblast proliferation, differentiation, and integration in modified and un-modified gelatine sponges. Three scaffolds were studied: gelatine sponge (Gelfoam), gelatin sponge/mineral (hydroxyapatite) composite, and gelatin sponge/polymer (poly-lactide-co-glycolide) composite. 2-D plastic coverslip was used as control. The gelatin sponges were modified using PLGA coating and mineral deposition to increase biodegradation resistance and osteoblast proliferation respectively. The scaffolds were characterized using Scanning Electron Microscopy (SEM) and X-ray diffraction. Cell number (DNA content), cell-replication rate (thymidine assay), and cell differentiation (alkaline phosphatase activity) were measured 24 h, 3 days, and 1, 2, 3 weeks after the osteoblast-like cells were cultured onto the scaffolds. Cell penetration into the sponges was determined using haematoxylin-eosin staining. Both modified and unmodified gelatine sponges demonstrated ability to support cell growth and cells were able to penetrate into the sponge pores. In a comparison of different scaffolds, cell number and cell replication were highest in sponge/hydroxyapatite composite and lowest in sponge/PLGA composite.     

Development,optimization and characterization of a full-thickness tissue engineered human oral mucosal model for biological assessment of dental biomaterials

Restorative dental materials and oral health care products come into direct contact with oral mucosa and can cause adverse reactions. In order to obtain an accurate risk assessment, the in vitro test model must reflect the clinical situation as closely as possible. The aim of this study was to develop and optimize a three-dimensional full-thickness engineered human oral mucosal model, which can be used for biological assessment of dental materials. In this study human oral fibroblasts and keratinocytes were isolated from patients and seeded onto a number of collagen-based and synthetic scaffolds using a variety of cell seeding techniques and grown at the air/liquid interface to construct human oral mucosa equivalents. Suitability of 10 different scaffolds for engineering human oral mucosa was evaluated in terms of biocompatibility, biostability, porosity, and the ability to mimic normal human oral mucosa morphology. Finally an optimized full-thickness engineered human oral mucosa was developed and characterized using transmission electron microscopy and immunostaining. The oral mucosa reconstruct resembled native human oral mucosa and it has the potential to be used as an accurate and reproducible test model in mucotoxicity and biocompatibility evaluation of dental materials.     

Disordered Graphene/Quartz Fabric as Biocompatible and Conductive Scaffold Promising for Regulated Growth and Differentiation of Nerve Cells

Nowadays, the use of topographical features and electrical conductivity of scaffolds at the cell-substrate interface for effectively regulating cell growth and differentiation have gained increasing attention due to great demands for tissue engineering. Herein, a facile approach to the growth of highly disordered graphene nanosheets (HDGNs) is demonstrated on a cheap and weaving quartz-braided structure as a functionalized scaffold for the differentiation of nerve cells. The patterned aligned structure can effectively integrate the advantages of a conductive graphene-functional interface (favorable for cell attachment and growth), topologically woven surface structure, providing a flexible and multifunctional regulatory platform for nerve cell growth. Compared with monocrystal polycrystalline graphene, amorphous graphene has high biocompatibility due to sufficient active sites, and has high conductivity to the composite nonconductive substrate, which can realize electrical stimulation (ES) of cell differentiation. Herein, the HDGN/quartz fabric with high biocompatibility (the cell viability is 98%), and great electrical conductivity, is proved. Then, the applied ES coupled with HDGN/quartz fabric significantly enhances selective neuronal differentiation into neurons (the differentiation growth rate is 131%). Collectively, herein, a new material basis is provided for electric induction of cell growth and differentiation, providing more possibilities for the development of intelligent biological applications.     

Injectable and Crosslinkable PLGA‐Based Microribbons as 3D Macroporous Stem Cell Niche

Poly(lactide‐co‐glycolide) (PLGA) has been widely used as a tissue engineering scaffold. However, conventional PLGA scaffolds are not injectable, and do not support direct cell encapsulation, leading to poor cell distribution in 3D. Here, a method for fabricating injectable and intercrosslinkable PLGA microribbon‐based macroporous scaffolds as 3D stem cell niche is reported. PLGA is first fabricated into microribbon‐shape building blocks with tunable width using microcontact printing, then coated with fibrinogen to enhance solubility and injectability using aqueous solution. Upon mixing with thrombin, firbornogen‐coated PLGA microribbons can intercrosslink into 3D scaffolds. When subject to cyclic compression, PLGA microribbon scaffolds exhibit great shock‐absorbing capacity and return to their original shape, while conventional PLGA scaffolds exhibit permanent deformation after one cycle. Using human mesenchymal stem cells (hMSCs) as a model cell type, it is demonstrated that PLGA μRB scaffolds support homogeneous cell encapsulation, and robust cell spreading and proliferation in 3D. After 28 days of culture in osteogenic medium, hMSC‐seeded PLGA μRB scaffolds exhibit an increase in compressive modulus and robust bone formation as shown by staining of alkaline phosphatase, mineralization, and collagen. Together, the results validate PLGA μRBs as a promising injectable, macroporous, non‐hydrogel‐based scaffold for cell delivery and tissue regeneration applications.     

Three dimensional printed macroporous polylactic acid/hydroxyapatite composite scaffolds for promoting bone formation in a critical-size rat calvarial defect model

We have explored the applicability of printed scaffold by comparing osteogenic ability and biodegradation property of three resorbable biomaterials. A polylactic acid/hydroxyapatite (PLA/HA) composite with a pore size of 500 μm and 60% porosity was fabricated by three-dimensional printing. Three-dimensional printed PLA/HA, β-tricalcium phosphate (β-TCP) and partially demineralized bone matrix (DBM) seeded with bone marrow stromal cells (BMSCs) were evaluated by cell adhesion, proliferation, alkaline phosphatase activity and osteogenic gene expression of osteopontin (OPN) and collagen type I (COL-1). Moreover, the biocompatibility, bone repairing capacity and degradation in three different bone substitute materials were estimated using a critical-size rat calvarial defect model in vivo. The defects were evaluated by micro-computed tomography and histological analysis at four and eight weeks after surgery, respectively. The results showed that each of the studied scaffolds had its own specific merits and drawbacks. Three-dimensional printed PLA/HA scaffolds possessed good biocompatibility and stimulated BMSC cell proliferation and differentiation to osteogenic cells. The outcomes in vivo revealed that 3D printed PLA/HA scaffolds had good osteogenic capability and biodegradation activity with no difference in inflammation reaction. Therefore, 3D printed PLA/HA scaffolds have potential applications in bone tissue engineering and may be used as graft substitutes in reconstructive surgery.     

ZnO/Nanocarbons‐Modified Fibrous Scaffolds for Stem Cell‐Based Osteogenic Differentiation

Currently, mesenchymal stem cells (MSCs)‐based therapies for bone regeneration and treatments have gained significant attention in clinical research. Though many chemical and physical cues which influence the osteogenic differentiation of MSCs have been explored, scaffolds combining the benefits of Zn²⁺ ions and unique nanostructures may become an ideal interface to enhance osteogenic and anti‐infective capabilities simultaneously. In this work, motivated by the enormous advantages of Zn‐based metal–organic framework‐derived nanocarbons, C‐ZnO nanocarbons‐modified fibrous scaffolds for stem cell‐based osteogenic differentiation are constructed. The modified scaffolds show enhanced expression of alkaline phosphatase, bone sialoprotein, vinculin, and a larger cell spreading area. Meanwhile, the caging of ZnO nanoparticles can allow the slow release of Zn²⁺ ions, which not only activate various signaling pathways to guide osteogenic differentiation but also prevent the potential bacterial infection of implantable scaffolds. Overall, this study may provide new insight for designing stem cell‐based nanostructured fibrous scaffolds with simultaneously enhanced osteogenic and anti‐infective capabilities.     

Novel 3D porous multi-phase composite scaffolds based on PCL,thermoplastic zein and ha prepared via supercritical CO2 foaming for bone regeneration

The aim of this study was the design of novel biodegradable porous scaffolds for bone tissue engineering (bTE) via supercritical CO₂ (scCO₂) foaming process. The porous scaffolds were prepared from a poly(ε-caprolactone)-thermoplastic zein multi-phase blend w/o interdispersed hydroxyapatite particles (HA) and the porous structure achieved via the scCO₂ foaming technology. The control of scaffolds porosity was obtained by modulating materials formulation and foaming temperature (T_F). The scaffolds were subjected to morphological, micro-structural and biodegradation analyses, as well as in vitro biocompatibility tests. Results demonstrated that both HA concentration and T_F significantly affected the micro-structural features of the scaffolds. In particular, scaffolds with porosity and pore size distribution, mechanical properties and biodegradability adequate for bTE were designed and produced by selecting a T_F equal to 100 °C for all the compositions used. The biocompatibility of these scaffolds was assessed in vitro by using osteoblast-like MG63 and human mesenchymal stem cells (hMSCs).     

Stem cell homing-based tissue engineering using bioactive materials

Tissue engineering focuses on repairing tissue and restoring tissue functions by employing three elements: scaffolds, cells and biochemical signals. In tissue engineering, bioactive material scaffolds have been used to cure tissue and organ defects with stem cell-based therapies being one of the best documented approaches. In the review, different biomaterials which are used in several methods to fabricate tissue engineering scaffolds were explained and show good properties (biocompatibility, biodegradability, and mechanical properties etc.) for cell migration and infiltration. Stem cell homing is a recruitment process for inducing the migration of the systemically transplanted cells, or host cells, to defect sites. The mechanisms and modes of stem cell homing-based tissue engineering can be divided into two types depending on the source of the stem cells: endogenous and exogenous. Exogenous stem cell-based bioactive scaffolds have the challenge of long-term culturing in vitro and for endogenous stem cells the biochemical signal homing recruitment mechanism is not clear yet. Although the stem cell homing-based bioactive scaffolds are attractive candidates for tissue defect therapies, based on in vitro studies and animal tests, there is still a long way before clinical application.     

Tissue Mimicry in Morphology and Composition Promotes Hierarchical Matrix Remodeling of Invading Stem Cells in Osteochondral and Meniscus Scaffolds

An integral approach toward in situ tissue engineering through scaffolds that mimic tissue with regard to both tissue architecture and biochemical composition is presented. Monolithic osteochondral and meniscus scaffolds are prepared with tissue analog layered biochemical composition and perpendicularly oriented continuous micropores by a newly developed cryostructuring technology. These scaffolds enable rapid cell ingrowth and induce zonal‐specific matrix synthesis of human multipotent mesenchymal stromal cells solely through their design without the need for supplementation of soluble factors such as growth factors.     

In Vitro Characterizations of PLLA/β-TCP Porous Matrix Materials and RMSC-PLLA-β-TCP Composite Scaffolds

To develop a novel degradable poly (L-lactic acid)/β-tricalcium phosphate (PLLA/β-TCP) bioactive materials for bone tissueengineering, β-TCP powder was produced by a new wet process. Porous scaffolds were prepared by three steps, i.e. solventcasting, compression molding and leaching stage. Factors influencing the compressive strength and the degradation behaviorof the porous scaffold, e.g. weight fraction of pore forming agent-sodium chloride (NaCl), weight ratio of PLLA: β-TCP,the particle size of β-TCP and the porosity, were discussed in details. Rat marrow stromal cells (RMSC) were incorporatedinto the composite by tissue engineering approach. Biological and osteogenesis potential of the composite scaffold weredetermined with MTT assay, alkaline phosphatase (ALP) activity and bone osteocalcin (OCN) content evaluation. Resultsshow that PLLA/β-TCP bioactive porous scaffold has good mechanical and pore structure with adjustable compressive strengthneeded for surgery. RMSCs seeding on porous PLLA/