A tendon is a fibrous connective tissue that acts to transmit tensile forces between muscles and bones. It mainly consists of soluble substance, collagen and small volume of elastic fibres, which are produced by tenoblasts and tenocytes. The Achilles tendon is the thickest tendon in the human body that subjects to some of the highest tensile force, thus disorders and ruptures commonly happen. As the insoluble fibrous components in Achilles tendons, the collagen fibrils and elastic fibres have unique spatial structure that plays important functional roles. Despite this, the understanding of relationship between them is still limited due to the lack of imaging evidence. Using confocal and second harmonic generation microscopy, this study aims to comprehensively investigate the spatial relationship of collagen, elastic fibres and tenocytes in hydrated tendons.

Longitudinal sections of 50 µm thick and transverse sections of 20 µm thick were cryo-sectioned respectively from the mid-portion of ten rabbit Achilles tendons. Sections were stained with 0.03g/L Acridine Orange (AO) and 1mg/ml Sulforhodamine B (SRB) solution respectively for labelling the nucleus and elastic fibres. The Leica TCS SP2 multiphoton microscopy containing second harmonic generation microscopy can image collagen without labelling. The sections were scanned by the multiphoton microscopy, and images were processed and reconstructed into 3D images to study the spatial structure of collagen, elastic fibres and cells in Achilles tendons

A rabbit Achilles tendon consists of three sub-tendons named flexor digitorum superficialis tendon, medial gastrocnemius tendon and lateral gastrocnemius tendon. Loose connective tissue connects the three sub-tendons and ensures efficient sliding between sub-tendons. The 3D network shows that the mid-portion of Achilles tendons is composed of longitudinal collagen and elastic fibres, while spindle tenocytes rest along the collagen and elastic fibres. Tenocytes appear to have a closer microstructural relationship with the elastic fibres. In comparison with the collagen, tenocytes and elastic fibres only occupy a very small volume in the 3D network. The elastic fibres exist in both tendon proper and endotenons. The tendon sheath and loose connective tissue have a higher cell density, and the cells are large and round while compared with tenocytes.

As a component of the extracellular matrix (ECM) in Achilles tendons that closely mediates with the tenocytes, the elastin may participate in the force transition and interaction between tenocytes and the ECM. The elastic fibres may also endow Achilles tendons with unique mechanical properties to stand for tensile force.

Purpose: The current clinical treatment protocol for bone healing applies super-physiological dose of rhBMP7. Unfortunately, it may result in adverse side effects. Some studies have demonstrated a dose-dependent osteogenic differentiation using rodent bone marrow derived stem cells (BMSCs). However, the dose effect of BMP7 on osteogenic differentiation of normal human BMSCs is largely unknown. In the present study, we investigated in vitro osteogenic differentiation of hBMSCs with a gradient concentration of rhBMP7. The interaction between rhBMP7 and osteogenic differentiation medium (ODM) was also examined.

Method: The primary BMSCs from human bone marrow were cultured and maintained in MSC growth medium (MGM). Six study groups were designed: MGM only, MGM with rhBMP7 of 0.1ug/ml, ODM only, and ODM with 3 concentration of rhBMP7 of 0.01μg/ml, 0.1μg/ml, and 1.0μg/ml, respectively. Alkaline phosphatase level (ALP) at day 17 and cumulative calcium deposit at both day 17 and day 35 were examined. mRNA expression level of osteogenic markers including osteocalcin (OC), osteopontin (OPN) and ALP were quantified using real-time RT-PCR at day 17.

Results: ALP activity at day17 did not increase in MGM with or without 0.1μg/ml of rhBMP7, ODM alone and ODM with 0.01μg/ml of rhBMP7. ALP activity was much higher with 0.1μg/ml of rhBMP7 plus ODM (0.22±0.02IU) than that in MGM with 0.1μg/ml of rhBMP7 (0.01±0.01IU, P< 0.05).

Conclusion: Our study demonstrated that rhBMP7 induced osteogenic differentiation of hBMSCs in a dose-dependent manner in the presence of ODM and the minimal dose for inducing in vitro osteoblastic differentiation was 0.1ug/ml of rhBMP7 under synergistic effect of ODM. The results of this study provide some insights into further investigation of synergy of rhBMP7 with other molecules. The types and amounts of simple molecules could significantly reduce therapeutic dose of rhBMP7 and achieve equivalent or better outcomes in clinical application warrant further investigation.