Preparation of Type II Collagen Short Peptides: Temperature Conditions of Cartilage Homogenization and Collagen Hydrolysis.

Physicochemical properties of hyaline cartilage homogenates were studied by the method of microcalorimetry. Collagen hydrolysates were obtained after homogenization of hyaline cartilages under high pressure conditions at the temperatures that denaturate collagen. Thermodynamic parameters of thermal transition of collagen in cartilage suspension were determined. Enthalpy of thermal transition ΔН decreases in comparison with the control. Thermal transition half-width ΔТ varies with temperature. More denatured and homogeneous samples were obtained at homogenization temperature 80°C. According to spectral studies, particles in the samples obtained at the temperature of 80°C were smaller. The temperature of 80°C is preferred for homogenizing hyaline cartilages and obtaining collagen type II short peptides.

Bionic composite hydrogel with a hybrid covalent/noncovalent network promoting phenotypic maintenance of hyaline cartilage.

The injectable composite hydrogel based on collagen and hyaluronic acid provided a bionic three-dimensional microenvironment and mimetic natural extracellular matrix (ECM) for the growth of cells in vivo and has been widely researched and developed for cartilage tissue engineering. Here, a novel injectable bionic hydrogel with hybrid covalent/noncovalent network derived from covalent conjugation of HA-SH and noncovalent supramolecular self-assembly of BPAA-AFF-OH short peptide was fabricated to overcome the collagen immunogenicity of animal origin and effectively maintain its biological function. Moreover, through optimizing the network structure and polymer composition, the bionic HS5FFAB5 hydrogel presented a reliable mechanical strength which depended on the highly integrated fiber structure between HA-SH and FFAB-AFF-OH molecules. The results in vitro and in vivo proved that HA-SH could provide a fundamental frame structure, while the supramolecular hydrogels could reinforce this structure via hydrogen bonds and hydrophilic/hydrophobic interactions, and endow bionic hydrogels with more abundant cell adhesion sites. The bionic composite hydrogel could improve the cell adhesion and proliferation when compared to HA-SH hydrogel, and enhanced chondrogenic related gene expression and matrix secretion by three-dimensional co-cultured in vitro and subcutaneous implantation in vivo, which further promoted phenotypic maintenance of hyaline cartilage. This bionic hydrogel with a hybrid covalent/noncovalent network is supposed to have potential application prospects in cartilage regeneration.

Glucosamine-grafted methacrylated gelatin hydrogels as potential biomaterials for cartilage repair.

Glucosamine (GlcN) has been widely used to reduce joint pain and osteoarthritis progression, but the efficacy of GlcN remains controversial because of the low GlcN concentration reaching the articular cavity. The aim of this study is to provide an effective approach of GlcN delivery to a target site using photocrosslinkable methacrylated gelatin (GelMA)-based hydrogels, where GlcN could be gradually released during the degradation of the GelMA hydrogel. Herein, GlcN was acrylated as the acryloyl glucosamine (AGA) and covalently grafted to GelMA, and more than 87.7% of 15% (w/v) GelMA hydrogel was grafted with AGA. Moreover, in vitro outgrowth and apoptosis assay of bone marrow stem cells (BMSCs) demonstrated that the GelMA-AGA hydrogels had better biocompatibility, larger cell attachment, and higher cell viability than pure GlcN and AGA materials. Also, 15% (w/v) GelMA-AGA hydrogel was injected into the defect site for in vivo rabbit cartilage repair. Compared with oral administration of pure GlcN and injection of pure GelMA, the repaired cartilages using GelMA-AGA hydrogels had the smoothest surface of the defect site, filling more than 95% defect bulk. The similar content of glycosaminoglycans to the native tissue and the maximum amount of type II collagen was found in the repaired cartilage using GelMA-AGA hydrogels, indicating the outgrowth of hyaline cartilage.

Glutamic acid-based dendritic peptides for scaffold-free cartilage tissue engineering.

Current treatment modalities for cartilage regeneration often result in the production of fibrous-type cartilage tissue at the defect site, which has inferior mechanical properties as compared to native hyaline cartilage. Further, effective treatments are not available at present, for preventing age-related as well as disease-related hypertrophic development of chondrocytes. In the present study, we designed and synthesized three sets of glutamic acid-based dendritic peptides, differing in degree of lipidation as well as branching. Each set constitutes of N-terminal protected as well as corresponding N-deprotected peptides. Altogether, six peptides [BE12, E12, BE3(12)4, E3(12)4, BE3OMe, E3OMe] were tested for their chondrogenesis enhancing potential in vitro, using rabbit adipose derived mesenchymal stem cells (ADMSCs). Immunohistochemical and gene expression studies as well as biochemical analyses revealed that the lipopeptides [E12 and BE3(12)4] are able to enhance chondrogenic differentiation of ADMSCs significantly (p < 0.001) as compared to control group (chondrogenic medium alone). Glycosaminoglycan content, and the chondrogenic marker genes like Aggrecan (Acan), Type II collagen (Col2a1), Hyaluronan synthase 2 (Has2), and SRY-box 9 (Sox9) expressions were found to be significantly increased in E12 and BE3(12)4 treated groups. Most importantly, the BE3(12)4 treated group showed significantly lower Type I collagen (Col1a2) and Type X collagen (Col10a1) transcript levels (p < 0.001), indicating its potential for hyaline cartilage formation and also to prevent hypertrophic development. Thus, the lipopeptides E12 and BE3(12)4 may be useful for preventing chondrocyte hypertrophy and realizing the hyaline nature of regenerated cartilage tissue in tissue engineering. STATEMENT OF SIGNIFICANCE: The current treatment modalities for degenerative cartilage diseases are unsatisfactory as the resultant regenerated cartilage is often fibrous in nature with inferior mechanical properties. Further, there is no proper treatment available for age-related development of chondrocyte hypertrophy at present. In this study we synthesized glutamic acid-based lipopeptides, which differ in the degree of lipidation as well as branching. We used a combinatorial approach of scaffold-free tissue engineering and dendritic lipopeptides to achieve hyaline-like cartilage tissue from adipose derived mesenchymal stem cells in vitro. Gene expression analysis revealed the down regulation of fibrous cartilage marker Col1a2 and hypertrophic marker Col10a1, suggesting that these lipopeptides may be useful for achieving mechanically superior hyaline cartilage regeneration in future.

Collagen Peptides from Hyaline Cartilage for the Treatment and Prevention of Joint Diseases: Isolation and Characteristics.

We studied the effects of Chymopsin and Caripazim on the proteolysis of collagen proteins from cattle tracheal hyaline cartilage. Homogenization of the cartilage under conditions of high pressure and temperature facilitated subsequent enzymatic hydrolysis: the degree of hydrolysis increased upon elevation of pressure from 40 to 80 mPa and temperature from 60 to 70°C. Proteolysis with Chymopsin yielded collagen peptides with molecular weights from 900 to 7000 Da, while Caripazim processing yielded collagen peptides with lower molecular weights from 250 to 780 Da consisting of 2-8 amino acids, which could be easily absorbed and intensely incorporated in the formation of the joint tissue structures.

Isolation and Characterisation of Major and Minor Collagens from Hyaline Cartilage of Hoki (Macruronus novaezelandiae).

The composition and properties of collagen in teleost (bony fish) cartilage have never been studied. In this study, we aimed to identify and characterise all collagen species in the nasal cartilage of hoki (Macruronus novaezelandiae). Four native collagen species were extracted using two techniques, and isolated with differential salt precipitation. We were able to assign the identity of three of these collagen species on the basis of solubility, SDS-PAGE and amino acid analyses. We found that hoki cartilage contains the major collagen, type II, and the minor collagens, type IX and type XI, which are homologous to those found in mammal and chicken cartilage. Using these extraction protocols, we also isolated a full-length type IX collagen from cartilage for the first time. In addition, we detected a 90 kDa, highly glycosylated collagen that has not been identified in any other species. For each isolate, structural and biochemical characterisations were performed using circular dichroism and Fourier transform infrared spectroscopy analyses, and the thermal denaturation properties were determined. Our results showed that the properties of hoki cartilage-derived collagens are similar to those of collagens in mammalian cartilage, indicating that teleost cartilage could provide biological ingredients for the development of biomaterials to treat cartilage-related illnesses.

Development of Low-Molecular Weight Collagen Peptide Complex with Glycosaminoglycan Components.

Enzymatic hydrolysis of biopolymers of the cartilage tissue was studied for obtaining a complex of type II collagen peptides and glycosaminoglycan oligosaccharides. Hydrothermal hydrolysis in a high pressure homogenizer followed by enzymatic hydrolysis of the cartilage tissue biopolymers with proteolytic enzyme preparation Karipazim yielded a complex of collagen peptides and glycosaminoglycan oligosaccharides with molecular weights of 240-720 Da. Low molecular weight of the components increases their bioavailability. Entering into the cells (chondrocytes), low-molecular-weight peptides, disaccharides, and oligosaccharides as structural elements of the matrix can participate in the formation of fibrils of collagen and proteoglycans. Exogenous substances replenish deficient components of the matrix and/or their concentrations, affect the formation and strengthen the cartilage tissue. Thus, using cattle and porcine hyaline cartilages, we prepared a complex of biopolymers with lower molecular weights in comparison with previously developed nutraceuticals.

Quantifying Baseline Fixed Charge Density in Healthy Human Cartilage Endplate: A Two-point Electrical Conductivity Method.

STUDY DESIGN: Regional measurements of fixed charge densities (FCDs) of healthy human cartilage endplate (CEP) using a two-point electrical conductivity approach. OBJECTIVE: The aim of this study was to determine the FCDs at four different regions (central, lateral, anterior, and posterior) of human CEP, and correlate the FCDs with tissue biochemical composition. SUMMARY OF BACKGROUND DATA: The CEP, a thin layer of hyaline cartilage on the cranial and caudal surfaces of the intervertebral disc, plays an irreplaceable role in maintaining the unique physiological mechano-electrochemical environment inside the disc. FCD, arising from the carboxyl and sulfate groups of the glycosaminoglycans (GAG) in the extracellular matrix of the disc, is a key regulator of the disc ionic and osmotic environment through physicochemical and electrokinetic effects. Although FCDs in the annulus fibrosus (AF) and nucleus pulposus (NP) have been reported, quantitative baseline FCD in healthy human CEP has not been reported. METHODS: CEP specimens were regionally isolated from human lumbar spines. FCD and ion diffusivity were concurrently investigated using a two-point electrical conductivity method. Biochemical assays were used to quantify regional GAG and water content. RESULTS: FCD in healthy human CEP was region-dependent, with FCD lowest in the lateral region (P = 0.044). Cross-region FCD was 30% to 60% smaller than FCD in NP, but similar to the AF and articular cartilage (AC). CEP FCD (average: 0.12 ±â€Š0.03 mEq/g wet tissue) was correlated with GAG content (average: 31.24 ±â€Š5.06 µg/mg wet tissue) (P = 0.005). In addition, the cross-region ion diffusivity in healthy CEP (2.97 ±â€Š1.00 × 10 cm/s) was much smaller than the AF and NP. CONCLUSION: Healthy human CEP acts as a biomechanical interface, distributing loads between the bony vertebral body and soft disc tissues and as a gateway impeding rapid solute diffusion through the disc. LEVEL OF EVIDENCE: N/A.

Mechano growth factor (MGF) and transforming growth factor (TGF)-ß3 functionalized silk scaffolds enhance articular hyaline cartilage regeneration in rabbit model.

Damaged cartilage has poor self-healing ability and usually progresses to scar or fibrocartilaginous tissue, and finally degenerates to osteoarthritis (OA). Here we demonstrated that one of alternative isoforms of IGF-1, mechano growth factor (MGF) acted synergistically with transforming growth factor ß3 (TGF-ß3) embedded in silk fibroin scaffolds to induce chemotactic homing and chondrogenic differentiation of mesenchymal stem cells (MSCs). Combination of MGF and TGF-ß3 significantly increased cell recruitment up to 1.8 times and 2 times higher than TGF-ß3 did in vitro and in vivo. Moreover, MGF increased Collagen II and aggrecan secretion of TGF-ß3 induced hMSCs chondrogenesis, but decreased Collagen I in vitro. Silk fibroin (SF) scaffolds have been widely used for tissue engineering, and we showed that methanol treated pured SF scaffolds were porous, similar to compressive module of native cartilage, slow degradation rate and excellent drug released curves. At 7 days after subcutaneous implantation, TGF-ß3 and MGF functionalized silk fibroin scaffolds (STM) recruited more CD29+/CD44+cells (P<0.05). Similarly, more cartilage-like extracellular matrix and less fibrillar collagen were detected in STM scaffolds than that in TGF-ß3 modified scaffolds (ST) at 2 months after subcutaneous implantation. When implanted into articular joints in a rabbit osteochondral defect model, STM scaffolds showed the best integration into host tissues, similar architecture and collagen organization to native hyaline cartilage, as evidenced by immunostaining of aggrecan, collagen II and collagen I, as well as Safranin O and Masson's trichrome staining, and histological evalution based on the modified O'Driscoll histological scoring system (P<0.05), indicating that MGF and TGF-ß3 might be a better candidate for cartilage regeneration. This study demonstrated that TGF-ß3 and MGF functionalized silk fibroin scaffolds enhanced endogenous stem cell recruitment and facilitated in situ articular cartilage regeneration, thus providing a novel strategy for cartilage repair.

Feasibility of high-resolution one-dimensional relaxation imaging at low magnetic field using a single-sided NMR scanner applied to articular cartilage.

Low field Nuclear Magnetic Resonance increases the contrast of the longitudinal relaxation rate in many biological tissues; one prominent example is hyaline articular cartilage. In order to take advantage of this increased contrast and to profile the depth-dependent variations, high resolution parameter measurements are carried out which can be of critical importance in an early diagnosis of cartilage diseases such as osteoarthritis. However, the maximum achievable spatial resolution of parameter profiles is limited by factors such as sensor geometry, sample curvature, and diffusion limitation. In this work, we report on high-resolution single-sided NMR scanner measurements with a commercial device, and quantify these limitations. The highest achievable spatial resolution on the used profiler, and the lateral dimension of the sensitive volume were determined. Since articular cartilage samples are usually bent, we also focus on averaging effects inside the horizontally aligned sensitive volume and their impact on the relaxation profiles. Taking these critical parameters into consideration, depth-dependent relaxation time profiles with the maximum achievable vertical resolution of 20 µm are discussed, and are correlated with diffusion coefficient profiles in hyaline articular cartilage in order to reconstruct T(2) maps from the diffusion-weighted CPMG decays of apparent relaxation rates.

Is the T1ρ MRI profile of hyaline cartilage in the normal hip uniform?

BACKGROUND: T1ρ MRI is an imaging technique sensitive to proteoglycan (PG) content of hyaline cartilage. However, normative T1ρ values have not been established for the weightbearing cartilage of the hip, and it is not known whether it is uniform or whether there is topographic variation. Knowledge of the T1ρ profile of hyaline cartilage in the normal hip is important for establishing a baseline against which comparisons can be made to experimental and clinical arthritic subjects. QUESTIONS/PURPOSES: In this diagnostic study, we determined (1) the T1ρ MRI values of hyaline cartilage of the normal hip; and (2) whether the T1ρ MRI profile of the normal hip hyaline cartilage is uniform. METHODS: Fourteen asymptomatic volunteers (11 men, three women; mean age, 35 years) prospectively underwent 1.5-T T1ρ MRI of a single hip. The weightbearing hyaline cartilage bilayer of the acetabulum and femoral head was evaluated on sagittal images and segmented into four zones: (1) anterior; (2) anterosuperior; (3) posterosuperior; and (4) and posterior. For the full region of interest and within each zone and each sagittal slice, we calculated the mean T1ρ relaxation value, a parameter that indirectly quantifies PG content, where T1ρ is inversely related to PG concentration. RESULTS: There was variation in the T1ρ relaxation values depending on zone (anterior to posterior) and slice (medial to lateral). When combining the most anterior quadrants (Zones 1 and 2), the T1ρ relaxation values were lower than those in the combined posterior quadrants (Zones 3 and 4) (30.4 msec versus 32.2 msec, respectively; p = 0.002), reflecting higher PG concentration. There was a difference between the T1ρ relaxation values of the sagittal slices (p = 0.038), most pronounced anteriorly in Zone 1 (26.6 msec, p = 0.001). With a selective combination of zones and slices, there were lower mean T1ρ values in the anterolateral-most region compared with the remainder of the weightbearing portion of the hip (28.6 msec versus 32.2 msec, respectively; p = 0.001). CONCLUSIONS: The T1ρ profile of normal hyaline cartilage of the hip is not uniform with the topographic differences identified suggesting regional variations in PG concentration. This study, through determination of lower T1ρ relaxation values, suggests inherently greater PG concentrations in the more anterolateral region of the normal hip hyaline cartilage. Furthermore, it demonstrates that T1ρ MRI has the ability to detect even subtle, microscopic local differences in hyaline cartilage composition. This technique has the potential to facilitate basic science and clinical research by serving as a noninvasive surrogate or biomarker of cartilage health and thus may be added to the growing repertoire of advanced, biochemical MRI techniques for evaluating hyaline cartilage.

Study of differential properties of fibrochondrocytes and hyaline chondrocytes in growing rabbits.

We aimed to build a culture model of chondrocytes in vitro, and to study the differential properties between fibrochondrocytes and hyaline chondrocytes. Histological sections were stained with haematoxylin and eosin so that we could analyse the histological structure of the fibrocartilage and hyaline cartilage. Condylar fibrochondrocytes and femoral hyaline chondrocytes were cultured from four, 4-week-old, New Zealand white rabbits. The production of COL2A1, COL1OA1, SOX9 and aggrecan was detected by real time-q polymerase chain reaction (RT-qPCR) and immunoblotting and the differences between them were compared statistically. Histological structures obviously differed between fibrocartilage and hyaline cartilage. COL2A1 and SOX9 were highly expressed within cell passage 2 (P2) of both fibrochondrocytes and hyaline chondrocytes, and reduced significantly after cell passage 4 (P4). The mRNA expressions of COL2A1 (p=0.05), COL10A1 (p=0.04), SOX9 (p=0.03), and aggrecan (p=0.04) were significantly higher in hyaline chondrocytes than in fibrochondrocytes, whereas the expression of COL1A1 (p=0.02) was the opposite. Immunoblotting showed similar results. We have built a simple and effective culture model of chondrocytes in vitro, and the P2 of chondrocytes is recommended for further studies. Condylar fibrocartilage and femoral hyaline cartilage have unique biological properties, and the regulatory mechanisms of endochondral ossification for the condyle should be studied independently in the future.

Assessment of hyaline cartilage matrix composition using near infrared spectroscopy.

Changes in the composition of the extracellular matrix (ECM) are characteristic of injury or disease in cartilage tissue. Various imaging modalities and biochemical techniques have been used to assess the changes in cartilage tissue but lack adequate sensitivity, or in the case of biochemical techniques, result in destruction of the sample. Fourier transform near infrared (FT-NIR) spectroscopy has shown promise for the study of cartilage composition. In the current study NIR spectroscopy was used to identify the contributions of individual components of cartilage in the NIR spectra by assessment of the major cartilage components, collagen and chondroitin sulfate, in pure component mixtures. The NIR spectra were obtained using homogenous pellets made by dilution with potassium bromide. A partial least squares (PLS) model was calculated to predict composition in bovine cartilage samples. Characteristic absorbance peaks between 4000 and 5000 cm(-1) could be attributed to components of cartilage, i.e. collagen and chondroitin sulfate. Prediction of the amount of collagen and chondroitin sulfate in tissues was possible within 8% (w/dw) of values obtained by gold standard biochemical assessment. These results support the use of NIR spectroscopy for in vitro and in vivo applications to assess matrix composition of cartilage tissues, especially when tissue destruction should be avoided.

MRI rotating frame relaxation measurements for articular cartilage assessment.

In the present work we introduced two MRI rotating frame relaxation methods, namely adiabatic T1ρ and Relaxation Along a Fictitious Field (RAFF), along with an inversion-prepared Magnetization Transfer (MT) protocol for assessment of articular cartilage. Given the inherent sensitivity of rotating frame relaxation methods to slow molecular motions that are relevant in cartilage, we hypothesized that adiabatic T1ρ and RAFF would have higher sensitivity to articular cartilage degradation as compared to laboratory frame T2 and MT. To test this hypothesis, a proteoglycan depletion model was used. Relaxation time measurements were performed at 0 and 48h in 10 bovine patellar specimens, 5 of which were treated with trypsin and 5 untreated controls were stored under identical conditions in isotonic saline for 48h. Relaxation times measured at 48h were longer than those measured at 0h in both groups. The changes in T2 and MT relaxation times after 48h were approximately 3 times larger in the trypsin treated specimens as compared to the untreated group, whereas increases of adiabatic T1ρ and RAFF were 4 to 5 fold larger. Overall, these findings demonstrate a higher sensitivity of adiabatic T1ρ and RAFF to the trypsin-induced changes in bovine patellar cartilage as compared to the commonly used T2 and MT. Since adiabatic T1ρ and RAFF are advantageous for human applications as compared to standard continuous-wave T1ρ methods, adiabatic T1ρ and RAFF are promising tools for assessing cartilage degradation in clinical settings.

Joint immobilization inhibits spontaneous hyaline cartilage regeneration induced by a novel double-network gel implantation.

We have recently discovered that spontaneous hyaline cartilage regeneration can be induced in an osteochondral defect in the rabbit, when we implant a novel double-network (DN) gel plug at the bottom of the defect. To clarify whether joint immobilization inhibits the spontaneous hyaline cartilage regeneration, we conducted this study with 20 rabbits. At 4 or 12 weeks after surgery, the defect in the mobile knees was filled with a sufficient volume of the hyaline cartilage tissue rich in proteoglycan and type-2 collagen, while no cartilage tissues were observed in the defect in the immobilized knees. Type-2 collagen, Aggrecan, and SOX9 mRNAs were expressed only in the mobile knees at each period. This study demonstrated that joint immobilization significantly inhibits the spontaneous hyaline cartilage regeneration induced by the DN gel implantation. This fact suggested that the mechanical environment is one of the significant factors to induce this phenomenon.

Hyaline cartilage surface study with an environmental scanning electron microscope. An experimental study.

To obtain images of the articular surface of fresh osteochondral grafts using an environmental scanning electron microscope (ESEM). To evaluate and compare the main morphological aspects of the chondral surface of the fresh grafts. To develop a validated classification system on the basis of the images obtained via the ESEM. The study was based on osteochondral fragments from the internal condyle of the knee joint of New Zealand rabbits, corresponding to fresh chondral surface. One hundred images were obtained via the ESEM and these were classified by two observers according to a category system. The Kappa index and the corresponding confidence interval (CI) were calculated. Of the samples analysed, 62-72% had an even surface. Among the samples with an uneven surface 17-22% had a hillocky appearance and 12-16% a knobbly appearance. As regards splits, these were not observed in 92-95% of the surfaces; 4-7% showed superficial splits and only 1% deep splits. In 78-82% of cases no lacunae in the surface were observed, while 17-20% showed filled lacunae and only 1-2% presented empty lacunae. The study demonstrates that the ESEM is useful for obtaining and classifying images of osteochondral grafts.

Immunolocalization of matrix proteins in different human cartilage subtypes.

Cartilage exerts many functions in different tissues and parts of the body. Specific requirements presumably also account for a specific biochemical composition. In this study, we investigated the presence and distribution pattern of matrix components, in particular collagen types in the major human cartilages (hyaline, fibrous, and elastic cartilage) by histochemical and immunohistochemical means. Macroscopically normal articular cartilages, menisci, disci (lumbar spine), epiglottal, and tracheal tissues were obtained from donors at autopsy. Aurical and nasal cartilages were part of routine biopsy samples from tumor resection specimens. Conventional histology and immunohistochemical stainings with collagen types I, II, III, IV, V, VI, and X and S-100 protein antibodies were performed on paraformaldehyde-fixed and paraffin-embedded specimens. The extracellular matrix is the functional component of all cartilages as indicated by the low cell densities. In particular major scaffold forming collagen types I (in fibrous cartilage) and II (in hyaline and elastic cartilages) as well as collagen type X (in the calcified layer of articular cartilages, the inner part of tracheal clips, and epiglottis cartilage) showed a specific distribution. In contrast, the "minor" collagen types III, V, and VI were found in all, collagen type IV in none of the cartilage subtypes. In this study, we present a biochemical profile of the major cartilage types of the human body which is important for understanding the physiology and the pathophysiology of cartilages.

Distinction between the extracellular matrix of the nucleus pulposus and hyaline cartilage: a requisite for tissue engineering of intervertebral disc.

Tissue engineering of intervertebral discs (IVD) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. However, since there is no unique marker characteristic of discs and since hyaline cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. This study was performed to compare the proteoglycan to collagen ratio (measured as GAG to hydroxyproline ratio) in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals and test the hypothesis that this ratio can be used for in vivo studies to distinguish between a normal NP and hyaline cartilage phenotype. Whole human lumbar spine specimens from fresh cadavers, ranging in age from 12 weeks to 79 years, were used to harvest the IVDs and adjacent endplates. The GAG to hydroxyproline ratio within the NP of young adults is approximately 27:1, whereas the ratio within the hyaline cartilage endplate of the same aged individuals is about 2:1. The production of an extracellular matrix with a high proteoglycan to collagen ratio can be used in vivo to distinguish NP cells from chondrocytes, and could help in identifying a NP-like phenotype in vivo as opposed to a chondrocyte when MSCs are induced to differentiate for tissue engineering of a disc.