Combined Application of Prototype Ultrasound and BSA-Loaded PLGA Particles for Protein Delivery.

PURPOSE: To develop an in vitro culture system for tissue engineering to mimic the in vivo environment and evaluate the applicability of ultrasound and PLGA particle system. METHODS: For tissue engineering, large molecules such as growth factors for cell differentiation should be supplied in a controlled manner into the culture system, and the in vivo microenvironment need to be reproduced in the system for the regulation of cellular function. In this study, portable prototype ultrasound with low intensity was devised and tested for protein release from bovine serum albumin (BSA)-loaded poly(lactic-co-glycolic acid) (PLGA) particles. RESULTS: BSA-loaded PLGA particles were prepared using various types of PLGA reagents and their physicochemical properties were characterized including particle size, shape, or aqueous wetting profiles. The BSA-loaded formulation showed nano-ranged size distribution with optimal physical stability during storage period, and protein release behaviors in a controlled manner. Notably, the application of prototype ultrasound with low intensity influenced protein release patterns in the culture system containing the BSA-loaded PLGA formulation. The results revealed that the portable ultrasound set controlled by the computer could contribute for the protein delivery in the culture medium. CONCLUSIONS: This study suggests that combined application with ultrasound and protein-loaded PLGA encapsulation system could be utilized to improve culture system for tissue engineering or cell regeneration therapy.

Synergistic Effect of Growth Factor Releasing Polymeric Nanoparticles and Ultrasound Stimulation on Osteogenic Differentiation.

Mesenchymal stem cells (MSCs) have been extensively used in the tissue regeneration therapy. Ex vivo therapy with well-differentiated osteogenic cells is known as an efficient treatment for musculoskeletal diseases, including rheumatoid diseases. However, along with its high cost, the current therapy has limitations in terms of restoring bone regeneration procedures. An efficient process for the cell differentiation to obtain a large number of functionalized osteogenic cells is necessary. Therefore, it is strongly recommended to develop strategies to produce sufficient numbers of well-differentiated osteogenic cells from the MSCs. In general, differentiation media with growth factors have been used to facilitate cell differentiation. In the present study, the poly (lactic-co-glycolic acid) (PLGA) nanoparticles incorporating the growth factors were included in the media, resulting in releasing growth factors (dexamethasone and ß-glycerophosphate) in the media in the controlled manner. Stable growth and early differentiation of osteogenic cells were achieved by the PLGA-based growth factor releasing system. Moreover, low intensity pulsed ultrasound was applied to this system to induce cell differentiation process. The results revealed that, as a biomarker at early stage of osteogenic cell differentiation, Lamin A/C nuclear protein was efficiently expressed in the cells growing in the presence of PLGA-based growth factor reservoirs and ultrasound. In conclusion, our results showed that the ultrasound stimulation combined with polymeric nanoparticles releasing growth factors could potentially induce osteogenic cell differentiation.

Mechanical stimuli enhance simultaneous differentiation into oesophageal cell lineages in a double-layered tubular scaffold.

The tissue-engineered oesophagus serves as an alternative and promising therapeutic approach for long-gap oesophageal replacement. This study proposes an advanced in vitro culture platform focused on construction of the oesophagus by combining an electrospun double-layered tubular scaffold, stem cells, biochemical reagents, and biomechanical factors. Human mesenchymal stem cells were seeded onto the inner and outer surfaces of the scaffold. Mechanical stimuli were applied with a hollow organ bioreactor along with different biochemical reagents inside and outside of the scaffold. Electrospun fibres in a tubular scaffold were found to be randomly and circumferentially oriented for the inner and outer surfaces, respectively. Amongst the two types of mechanical stimuli, the intermittent shear flow that can simultaneously cause circumferential stretching due to hydrostatic pressure, and shear stress caused by flow on the inner surface, was found to be more effective for simultaneous differentiation into epithelial and muscle lineage than steady shear flow. Under these conditions, the expression of epithelial markers on the inner surface was significantly observed, although it was minimal on the outer surface. Muscle differentiation showed the opposite expression pattern. Meanwhile, the mechanical tests showed that the strength of the scaffold was improved after incubation for 14 days. We have developed a potential platform for tissue-engineered oesophagus construction. Specifically, simultaneous differentiation into epithelial and muscle lineages can be achieved by utilizing the double-layered scaffold and appropriate mechanical stimulation.

The combined effects of hierarchical scaffolds and mechanical stimuli on ex vivo expansion of haematopoietic stem/progenitor cells.

We describe the ex vivo expansion of haematopoietic stem/progenitor cells (HSPCs) with consideration of their eventual in-vivo niche. We firstly fabricated hierarchically structured scaffolds (lattices derived via three-dimensional plotting combined with electrospun submicron fibers coated with vitronectin to increase cell affinity). We also applied intermittent hydrostatic pressure (IHP) to mimic the physical environment of the in vivo niche. In the absence of mechanical stimuli, the cell phenotype (CD34+, CD34+CD38-) remained excellent in the vitronectin-treated group. Two IHP regimens were tested; optimally, cells were pressurized (20 kPa) for 2 min and then rested for 13 min. On day 7 of culture, the total cell number had increased 21.2-fold and that of CD34+ cells 10.94-fold. CD34+ and CD34+CD38- cells constituted 44.50 and 44.07% of total cells, respectively. Colony-forming counts and the long-term culture-initiating cell assay showed that clonogenic potential was greatly improved under our experimental conditions. Scaffolds with hierarchical structures were valuable in this context. Furthermore, ex vivo expansion of HSPCs was improved by physical stimulation.

Combinational effects of mechanical forces and substrate surface characteristics on esophageal epithelial differentiation.

Even the efficacy of substrate and mechanical stimuli in addition to biochemical cues have been recognized in many studies of stem cell differentiation, few studies have been reported on the differentiation into esophageal epithelial cells. Therefore, the aim of this study was set to propose a method of differentiating stem cells into esophageal epithelial cells according to biochemical reagent concentration, substrate properties, and mechanical forces. After the concentration of all-trans retinoic acid was determined as 5 µM by a baseline experiment, the degree of differentiation was compared in three different kinds of substrates: cover glass, polyurethane (PU) membrane, and electrospun PU sheet (ePU). Then, on the substrate showing the more positive results, that is, ePU, two types of mechanical forces, intermittent hydrostatic pressure (IHP), and shear stress (SS), were applied individually at different magnitudes for the latter 7 days of an overall incubation period of 14 days. Following various biological assays, the lower IHP (50 mmHg) resulted in greater positive effects than the others. Even with cessation of the mechanical force, the relevant markers were remarkably increased. Although the range of factors regulating differentiation was limited, this study nonetheless demonstrated the combinational effects of mechanical force along with substrate type for the first time in related studies. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 552-560, 2019.

Cyclic stretch increases mitochondrial biogenesis in a cardiac cell line.

Unlike stable and immobile cell line conditions, animal hearts contract and relax to pump blood throughout the body. Mitochondria play an essential role by producing biological energy molecules to maintain heart function. In this study, we assessed the effect of heart mimetic cyclic stretch on mitochondria in a cardiac cell line. To mimic the geometric and biomechanical conditions surrounding cells in vivo, cyclic stretching was performed on HL-1 murine cardiomyocytes seeded onto an elastic micropatterned substrate (10% elongation, 0.5 Hz, 4 h/day). Cell viability, semi-quantitative Q-PCR, and western blot analyses were performed in non-stimulated control and cyclic stretch stimulated HL-1 cell lines. Cyclic stretch significantly increased the expression of mitochondria biogenesis-related genes (TUFM, TFAM, ERRα, and PGC1-α) and mitochondria oxidative phosphorylation-related genes (PHB1 and CYTB). Western blot analysis confirmed that cyclic stretch increased protein levels of mitochondria biogenesis-related proteins (TFAM, and ERRα) and oxidative phosphorylation-related proteins (NDUFS1, UQCRC, and PHB1). Consequently, cyclic stretch increased mitochondrial mass and ATP production in treated cells. Our results suggest that cyclic stretch transcriptionally enhanced mitochondria biogenesis and oxidative phosphorylation without detrimental effects in a cultured cardiac cell line.

Synergistic Integration of Mesenchymal Stem Cells and Hydrostatic Pressure in the Expansion and Maintenance of Human Hematopoietic/Progenitor Cells.

Ex vivo expansion of hematopoietic stem/progenitor cell (HSPC) has been investigated to improve the clinical outcome of HSPC transplantation. However, ex vivo expansion of HSPCs still faces a major obstacle in that HPSCs tend to differentiate when proliferating. Here, we cocultured HSPCs with mesenchymal stem cells (MSCs) and divided the HSPCs into two fractions according to whether they came into adherent to MSCs or not. Additionally, we used hydrostatic pressure (HP) to mimic the physical conditions in vivo. Even nonadherent cells expanded to yield a significantly larger number of total nucleated cells (TNCs), adherent cells maintained the HSPC phenotype (CD34+, CD34+CD38-, and CD133+CD38-) to a greater extent than nonadherent cells and had superior clonogenic potential. Moreover, applying HP significantly increased the number of TNCs, the frequency of the immature HSPC phenotype, and the clonogenic potential. Furthermore, the genetic markers for the HSPC niche were significantly increased under HP. Our data suggest that the nonadherent fraction is the predominant site of HSPC expansion, whereas the adherent fraction seems to mimic the HSPC niche for immature cells. Moreover, HP has a synergistic effect on expansion and functional maintenance. This first study utilizing HP has a potential of designing clinically applicable expansion systems.

Enhanced biocompatibility and osteogenic potential of mesoporous magnesium silicate/polycaprolactone/wheat protein composite scaffolds.

BACKGROUND: Successful bone tissue engineering using scaffolds is primarily dependent on the properties of the scaffold, including biocompatibility, highly interconnected porosity, and mechanical integrity. METHODS: In this study, we propose new composite scaffolds consisting of mesoporous magnesium silicate (m_MS), polycaprolactone (PCL), and wheat protein (WP) manufactured by a rapid prototyping technique to provide a micro/macro porous structure. Experimental groups were set based on the component ratio: (1) WP0% (m_MS:PCL:WP =30:70:0 weight per weight; w/w); (2) WP15% (m_MS:PCL:WP =30:55:15 w/w); (3) WP30% (m_MS:PCL:WP =30:40:30 w/w). RESULTS: Evaluation of the properties of fabricated scaffolds indicated that increasing the amount of WP improved the surface hydrophilicity and biodegradability of m_MS/PCL/WP composites, while reducing the mechanical strength. Moreover, experiments were performed to confirm the biocompatibility and osteogenic differentiation of human mesenchymal stem cells (MSCs) according to the component ratio of the scaffold. The results confirmed that the content of WP affects proliferation and osteogenic differentiation of MSCs. Based on the last day of the experiment, ie, the 14th day, the proliferation based on the amount of DNA was the best in the WP30% group, but all of the markers measured by PCR were the most expressed in the WP15% group. CONCLUSION: These results suggest that the m_MS/PCL/WP composite is a promising candidate for use as a scaffold in cell-based bone regeneration.

Characterization and osteogenic evaluation of mesoporous magnesium-calcium silicate/polycaprolactone/polybutylene succinate composite scaffolds fabricated by rapid prototyping.

The properties of scaffolds for bone tissue engineering, including their biocompatibility, highly interconnected porosity, and mechanical integrity, are critical for promoting cell adhesion, proliferation, and osteoinduction. We used various physical and biological assays to obtain in vitro confirmation that the proposed composite scaffolds are potentially suitable for applications to bone tissue engineering. The proposed new composite scaffolds, which we fabricated by a rapid prototyping technique, were composed of mesoporous magnesium-calcium silicate (m_MCS), polycaprolactone (PCL), and polybutylene succinate (PBSu). We systematically evaluated the characteristics of the composite scaffolds, such as the hydrophilicity and bioactivity. We also investigated the proliferation and osteogenic differentiation of human mesenchymal stem cells (MSCs) scaffolded on the m_MCS/PCL/PBSu composite. Our results showed that, compared to the m_MCS/PCL scaffold, the m_MCS/PCL/PBSu scaffold has improved water absorption, in vitro degradability, biocompatibility, and bioactivity in simulated body fluid, while its mechanical strength is reduced. Moreover, the results of the cytotoxicity tests specified in ISO 10993-12 and ISO 10993-5 clearly indicate that the m_MCS/PCL scaffold is not toxic to cells. In addition, we obtained significant increases in initial cell attachment and improvements to the osteogenic MSC differentiation by replacing the m_MCS/PCL scaffold with the m_MCS/PCL/PBSu scaffold. Our results indicate that the m_MCS/PCL/PBSu scaffold achieves enhanced bioactivity, degradability, cytocompatibility, and osteogenesis. As such, this scaffold is a potentially promising candidate for use in stem cell-based bone tissue engineering.

In vitro study on anti-inflammatory effects of epigallocatechin-3-gallate-loaded nano- and microscale particles.

PURPOSE: This study aimed to develop an anti-inflammation system consisting of epigallo-catechin-3-gallate (EGCG) encapsulated in poly(lactide-co-glycolic acid) (PLGA) particles to promote wound healing. METHODS: Nano- and microscale PLGA particles were fabricated using a water/oil/water emulsion solvent evaporation method. The optimal particle size was determined based on drug delivery efficiency and biocompatibility. The particles were loaded with EGCG. The anti-inflammatory effects of the particles were evaluated in an in vitro cell-based inflammation model. RESULTS: Nano- and microscale PLGA particles were produced. The microscale particles showed better biocompatibility than the nanoscale particles. In addition, the microscale particles released ~60% of the loaded drug, while the nanoscale particles released ~50%, within 48 hours. Thus, microscale particles were selected as the carriers. The optimal EGCG working concentration was determined based on the effects on cell viability and inflammation. A high EGCG dose (100 µM) resulted in poor cell viability; therefore, a lower dose (≤50 µM) was used. Moreover, 50 µM EGCG had a greater anti-inflammatory effect than 10 µM concentration on lipopolysaccharide-induced inflammation. Therefore, 50 µM EGCG was selected as the working dose. EGCG-loaded microparticles inhibited inflammation in human dermal fibroblasts. Interestingly, the inhibitory effects persisted after replacement of the drug-loaded particle suspension solution with fresh medium. CONCLUSION: The EGCG-loaded microscale particles are biocompatible and exert a sustained anti-inflammatory effect.

Effects of mechanical stimulation on the reprogramming of somatic cells into human-induced pluripotent stem cells.

BACKGROUND: Mechanical stimuli play important roles in the proliferation and differentiation of adult stem cells. However, few studies on their effects on induced pluripotent stem cells (iPSCs) have been published. METHODS: Human dermal fibroblasts were seeded onto flexible membrane-bottom plates, and infected with retrovirus expressing the four reprogramming factors OCT4, SOX2, KLF, and c-MYC (OSKM). The cells were subjected to equiaxial stretching (3% or 8% for 2, 4, or 7 days) and seeded on feeder cells (STO). The reprogramming into iPSCs was evaluated by the expression of pluripotent markers, in vitro differentiation into three germ layers, and teratoma formation. RESULTS: Equiaxial stretching enhanced reprogramming efficiency without affecting the viral transduction rate. iPSCs induced by transduction of four reprogramming factors and application of equiaxial stretching had characteristics typical of iPSCs in terms of pluripotency and differentiation potentials. CONCLUSIONS: This is the first study to show that mechanical stimuli can increase reprogramming efficiency. However, it did not enhance the infection rate, indicating that mechanical stimuli, defined as stretching in this study, have positive effects on reprogramming rather than on infection. Additional studies should evaluate the mechanism underlying the modulation of reprogramming of somatic cells into iPSCs.

Mechanical stimuli modulate intracellular calcium oscillations: a pathological model without chemical cues.

OBJECTIVE: To control the oscillatory behavior of the intracellular calcium ([Ca2+]i) concentration in endothelial cells via mechanical factors (i.e., various hydrostatic pressures) because [Ca2+]i in these cells is affected by blood pressure. RESULTS: Quantitative analyses based on real-time imaging showed that [Ca2+]i oscillation frequency and relative concentration increased significantly when 200 mm Hg pressure, mimicking hypertension, was applied for >10 min. Peak height and peak width decreased significantly at 200 mm Hg. These trends were more marked as the duration of the 200 mm Hg pressure was increased. However, no change was observed under normal blood pressure conditions 100 mm Hg. CONCLUSION: We generated a simple in vitro model to study [Ca2+]i behavior in relation to various pathologies and diseases by eliminating possible complicating effects induced by chemical cues.

The Effects of Epigallocatechin-3-Gallate and Mechanical Stimulation on Osteogenic Differentiation of Human Mesenchymal Stem Cells: Individual or Synergistic Effects.

This study aims to investigate the roles and effects of EGCG (epigallocatechin-3-gallate) during the osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro. Recent studies have shown that proper mechanical stimuli can induce osteogenic differentiation of hMSCs apart from biochemical factors. In this study, the hMSC cultures were subjected to: (1) 25 uM EGCG alone or (2) 3% mechanical stretching (0.2 Hz for 4 h/day for 4 days) or (3) in combination with 3% mechanical stretching (0.2 Hz for 4 h/day for 4 days). The two factors were applied to the cell cultures separately and in combination to investigate the individual and synergistic effect of both mechanical stimulation and ECGC in the osteogenic differentiation of hMSCs. Utilizing real time PCR, we measured various osteogenic markers and even those related to intracellular signalings. Further investigation of mitochondria was performed that mitochondria biogenesis, antioxidant capacity, and morphological related markers were measured. hMSCs were to be osteogenic or myogenic differentiated when they were under 3% stretching only. However, when EGCG was applied along with stretching they were to be osteogenic differentiated rather than to be myogenic differentiated. This was supported by evaluating intracellular signalings: BMP-2 and VEGF. Therefore, the synergistical effects of simultaneous employment of stretching and EGCG on osteogenic differentiation were confirmed. Moreover, simultaneous employment was found positive in mitochondria biogenesis, antioxidant capacity, and morphological changes. Through this study, we came into the conclusion that the combination of proper mechanical stretching, 3% in this study, and EGCG promote osteogenic differentiation. Reflecting that EGCG can be obtained from plants not from the chemical syntheses, it is worth to be studied further either by animal tests or long-term experiments for clinical applications.

PCL/ß-TCP Composite Scaffolds Exhibit Positive Osteogenic Differentiation with Mechanical Stimulation.

We investigated the use of Polycaprolactone (PCL)/ ß-tricalcium phosphate (ß-TCP) composites with applied mechanical stimulation as scaffold for bone tissue engineering. PCL-based three-dimensional (3D) structures were fabricated in a solvent-free process using a 3D-printing technique. The mass fraction of ß-TCP was varied in the range 0-30%, and the structure and compressive modulus of the specimens was characterized. The shape and interconnectivity of the pores was found to be satisfactory, and the compressive modulus of the specimens was comparable with that of human trabecular bone. Human mesenchymal stem cells were seeded on the composites, and various biological evaluations were performed over 9 days. With a mass fraction of ß-TCP of 30%, differentiation began earlier; however, the cell proliferation rate was lower. Through the use of mechanical stimulation, however, the proliferation rate recovered, and was comparable with that of the other groups. This stimulation effect was also observed in ECM generation and other biological assays. With mechanical stimulation, expression of osteogenic markers was lower on samples with a ß-TCP content of 10 wt% than without ß-TCP; however, with mechanical stimulation, the sample with a ß-TCP content of 30 wt% exhibited significantly greater expression of those markers than the other samples. We found that mechanical stimulation and the addition of ß-TCP interacted closely, and that a mass fraction of ß-TCP of 30% was particularly useful as a bone tissue scaffold when accompanied by mechanical stimulation.

Simultaneous engagement of mechanical stretching and surface pattern promotes cardiomyogenic differentiation of human mesenchymal stem cells.

It has been widely recognized and proved that biophysical factors for mimicking in vivo conditions should be also considered to have stem cells differentiated into desired cell type in vitro along with biochemical factors. Biophysical factors include substrate and biomechanical conditions. This study focused on the effect of biomimetic mechanical stretching along with changes in substrate topography to influence on cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs). Elastic micropatterned substrates were made to mimic the geometric conditions surrounding cells in vivo. To mimic biomechanical conditions due to beating of the heart, mechanical stretching was applied parallel to the direction of the pattern (10% elongation, 0.5 Hz, 4 h/day). Suberoylanilide hydroxamic acid (SAHA) was used as a biochemical factor. The micropatterned substrate was found more effective in the alignment of cytoskeleton and cardiomyogenic differentiation compared with flat substrate. Significantly higher expression levels of related markers [GATA binding protein 4 (GATA4), troponin I, troponin T, natriuretic peptide A (NPPA)] were observed when mechanical stretching was engaged on micropatterned substrate. In addition, 4 days of mechanical stretching was associated with higher levels of expression than 2 days of stretching. These results indicate that simultaneous engagement of biomimetic environment such as substrate pattern and mechanical stimuli effectively promotes the cardiomyogenic differentiation of hMSCs in vitro. The suggested method which tried to mimic in vivo microenvironment would provide systematic investigation to control cardiomyogenic differentiation of hMSCs.

Changes, and the Relevance Thereof, in Mitochondrial Morphology during Differentiation into Endothelial Cells.

The roles of mitochondria in various physiological functions of vascular endothelial cells have been investigated extensively. Morphological studies in relation to physiological functions have been performed. However, there have been few reports of morphological investigations related to stem cell differentiation. This was the first morphological study of mitochondria in relation to endothelial differentiation and focused on quantitative analysis of changes in mitochondrial morphology, number, area, and length during differentiation of human mesenchymal stem cells (hMSCs) into endothelial-like cells. To induce differentiation, we engaged vascular endothelial growth factors and flow-induced shear stress. Cells were classified according to the expression of von Willebrand factor as hMSCs, differentiating cells, and almost fully differentiated cells. Based on imaging analysis, we investigated changes in mitochondrial number, area, and length. In addition, mitochondrial networks were quantified on a single-mitochondrion basis by introducing a branch form factor. The data indicated that the mitochondrial number, area per cell, and length were decreased with differentiation. The mitochondrial morphology became simpler with progression of differentiation. These findings could be explained in view of energy level during differentiation; a higher level of energy is needed during differentiation, with larger numbers of mitochondria with branches. Application of this method to differentiation into other lineages will explain the energy levels required to control stem cell differentiation.

Shear stress and circumferential stretch by pulsatile flow direct vascular endothelial lineage commitment of mesenchymal stem cells in engineered blood vessels.

Understanding the response of mesenchymal stem cells (MSCs) in the dynamic biomechanical vascular environment is important for vascular regeneration. Native vessel biomechanical stimulation in vitro is thought to be the most important contributor to successful endothelial differentiation of MSCs. However, the appropriate biomechanical stimulation conditions for differentiating MSCs into ECs have not been fully investigated. To accomplish an in vivo-like loading environment, a loading system was designed to apply flow induced stress and induce hMSC differentiation in vascular cells. Culturing MSCs on tubular scaffolds under flow-induced shear stress (2.5 dyne/cm(2)) for 4 days results in increased mRNA levels of EC markers (vWF, CD31, VE-cadherin and E-selectin) after one day. Furthermore, we investigated the effects of 2.5 dyne/cm(2) shear stress followed by 3% circumferential stretch for 3 days, and an additional 5% circumferential stretch for 4 days on hMSC differentiation into ECs. EC marker protein levels showed a significant increase after applying 5% stretch, while SMC markers were not present at levels sufficient for detection. Our results demonstrate that the expression of several hMSC EC markers cultured on double-layered tubular scaffolds were upregulated at the mRNA and protein levels with the application of fluid shear stress and cyclic circumferential stretch.

A three-dimensional hierarchical scaffold fabricated by a combined rapid prototyping technique and electrospinning process to expand hematopoietic stem/progenitor cells.

OBJECTIVE: To investigate the expansion of hematopoietic stem/progenitor cells (HSPCs) from umbilical cord blood using extracellular matrix (ECM) protein-coated three-dimensional hierarchical scaffolds. RESULTS: The expansion of HSPCs was evaluated through total nucleated cell (TNC) expansion, immuno-phenotypic analysis, and clonogenic ability. After 7 days of culture, three-dimensional cultures with fibronectin-coated scaffolds achieved the highest fold increase in TNCs (164 ± 6.9 fold) and the highest CD45(+)CD34(+) (35 %) and CD34(+)CD38(-) (32 %) ratios. CONCLUSION: Three-dimensional hierarchical scaffolds were coated with ECM protein to simulate a biomimetic environment or niche, and had a significant effect on the expansion potential of HSPCs without changing their phenotype.

Distinguishing tendon and ligament fibroblasts based on 1H nuclear magnetic resonance spectroscopy.

Tendon and ligament (T/L) have been known to be obviously different from each other in tissue level. However, due to the overlapping gene markers, distinction in cellular level has not been clearly verified yet. Recently, the use of nuclear magnetic resonance (NMR) spectroscopy has shown the potential to detect biological markers in cellular level. Therefore, in this study we applied a non-invasive technique based on NMR spectroscopy to establish biomarkers to distinguish between T/L fibroblasts. In addition the cellular morphologies and gene expression patterns were also investigated for comparison through optical microscopy and real-time polymerase chain reaction (PCR). No difference was observed from morphology and real-time PCR results, either as expected. However, we found clear differences in their metabolomic spectra using 1H NMR spectroscopy. The calculated integral values of fatty acids (with chemical shifts at ~0.9, 1.26, 1.59, 2.05, 2.25, and 2.81 ppm), lactate (~1.33 ppm), and leucine (~2.72 ppm) were significantly different between the two types of fibroblasts. To be specific tendon group exhibited higher level of the metabolite than ligament group. In conclusion, in-cell metabolomic evaluation by NMR technique used in this study is believed to provide a promising tool in distinguishing cell types, especially T/L cells, which cannot be classified by conventional biological assays.