Human periodontal ligament stem cell seeding on calcium phosphate cement scaffold delivering metformin for bone tissue engineering

Abstract

Objectives

(1) develop a CPC-metformin scaffold with hPDLSC seeding for bone tissue engineering; and (2) investigate the effects of CPC-metformin scaffold on hPDLSC proliferation, osteogenic differentiation and bone matrix mineralization for the first time.

Methods

hPDLSCs were harvested from extracted teeth. CPC scaffolds (with or without metformin) were prepared. Three groups were tested: (1) control group (growth medium); (2) osteogenic group (osteogenic medium); (3) metformin + osteogenic group (CPC-metformin scaffold, cultured in osteogenic medium). hPDLSC viability, osteogenic differentiation and mineralization were measured. SEM was used to examine cell morphology.

Results

After culturing for 14 days, all three groups demonstrated excellent hPDLSC attachment and viability, as shown in live-dead staining, CCK-8 assay, and SEM examinations. The osteogenic group had 3–8 folds, 5 folds and 6 folds of increases in osteogenic gene expressions, ALP activity and mineral synthesis, compared to control group. Furthermore, the metformin + osteogenic group had 3-fold to 4-fold increases over those of the osteogenic group in osteogenic gene expressions, ALP activity and mineral synthesis.

Conclusions

hPDLSCs were demonstrated to be a potent cell source for bone engineering. The novel CPC-metformin-hPDLSC construct is highly promising to enhance bone repair and regeneration efficacy in dental, craniofacial and orthopedic applications.

Introduction

Bone defects often occur due to congenital defects, trauma, infection and other diseases that lead to the loss of bone tissues [1,2]. Due to an aging population, the need for bone repair and regeneration is rapidly growing. Although autografts are regarded as the gold-standard for repairing bone defects, their application is hindered by the invasive procedure and limited amount of bone harvest. Bone tissue engineering using stem cells and scaffolds is an exciting approach to meet this increasing need [3].

It is beneficial for the bone scaffold to mimic natural bone’s extracellular matrix and be able to induce and promote bone regeneration. Calcium phosphate cement (CPC) has excellent biocompatibility, osteoconductivity, osteoinductivity and bioactivity [[4], [5], [6]]. CPC is a promising scaffold material for dental and craniofacial repairs [6]. The CPC powder contains one or more kinds of calcium phosphate powders such as tetracalcium phosphate (TTCP) and dicalcium phosphate-anhydrous (DCPA). The CPC paste is formed by mixing the CPC powder with a liquid. The paste can be molded to the desired shape and then self-set and harden in situ to become a nanostructured and bone mineral-mimicking apatite scaffold in the bone defect [7].

Combining the scaffold with seed cells is promising to greatly enhance the tissue regeneration efficacy [[8], [9], [10]]. Three-dimensional scaffolds and cells can work synergistically to promote the tissue repair process [8,9,11,12]. The seed cells can enhance the bone tissue regeneration by differentiating into the osteogenic lineage and/or releasing molecules that increase the osteogenesis efficacy [7]. The most-frequently studied seed cells are human bone marrow mesenchymal stem cells (hBMSCs), which are regarded as the gold-standard cell source for bone tissue engineering research. However, hBMSCs require an invasive procedure to be harvested. Therefore, other stem cell sources are needed.

Human periodontal ligament stem cells (hPDLSCs) are mesenchymal stem cells (MSCs) isolated from the periodontal ligaments of extracted human teeth. Several studies revealed that hPDLSCs can be induced into the osteoblastic lineage, presenting osteogenic gene expressions and mineral synthesis [[13], [14], [15]]. hPDLSCs have several advantages, including the ease of harvesting from teeth that are extracted for reasons such as orthodontic treatments and removal of wisdom teeth, a strong self‐renewal ability, and multipotency characteristics [16]. hPDLSCs can differentiate into bone, muscle, nervous tissues, periodontium-like connective tissues and cementoid tissues [16,17]. Therefore, hPDLSCs are highly promising to serve as seed cells for periodontal tissue regeneration, as well as craniofacial and orthopedic tissue engineering applications. However, to date, there has been no report on the use of hPDLSCs with CPC scaffold for bone regeneration.

Metformin, an antidiabetic biguanide drug, was approved by the United States Food and Drug Administration (FDA) in 1995 for treating type 2 diabetes, and is currently widely used by diabetic patients worldwide [18]. Metformin is a non‐toxic and well-tolerated drug [19]. Intriguingly, recent studies indicated that metformin can also induce osteogenesis by promoting the differentiation of stem cells and preosteoblasts [20]. It was demonstrated that metformin enhanced the osteodifferentiation via the activation of the AMP-activated kinase (AMPK) signaling pathway [21]. Metformin showed the capability to increase the osteodifferentiation of several types of stem cells, including hBMSCs [22], human umbilical cord mesenchymal stem cells (hUMSCs) [23], human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC‐MSCs) [19], human dental pulp stem cells (hDPSCs) [24,25] and PDLSCs [26,27]. However, previous studies on the effects of metformin on hPDLSCs all directly added the metformin into the culture medium [26,27]. To facilitate potential clinical applications, it would be better to use a scaffold to deliver metformin which can be placed into a bone defect for local release. Our previous studies have shown that CPC scaffold containing chitosan is an effective delivery vehicle for metformin; however, hPDLSCs have not been seeded on CPC scaffold in previous studies [24,28]. To date, a literature search revealed no report on the combination of hPDLSCs with CPC-metformin scaffold for bone regeneration.

Therefore, the objectives of this study were to: (1) develop a CPC-metformin scaffold with hPDLSC seeding for bone tissue engineering; and (2) investigate the effects of CPC-metformin on hPDLSC proliferation, osteogenic differentiation and bone mineral synthesis for the first time. The following hypotheses were tested: (1) hPDLSCs seeded on CPC scaffold would be able to proliferate and successfully differentiate into the osteogenic lineage; (2) Compared to CPC scaffold without containing metformin, hPDLSCs seeded on CPC-metformin scaffold would have much greater osteogenic gene expressions, alkaline phosphatase activity and bone matrix mineral synthesis.