B1.Nowadays the development of a culturing technique to populate scaffolds is the greatest challenge in the field of tissue engineering. In this work, it will be shown how the spinner flask dynamic cell cultures should be used to populate hydroxyapatite microcarriers (HA-MC) for bone tissue engineering [B1].
B3 In bone tissue engineering, the most widely used scaffold materials is Calcium phosphate ceramics (e.g., hydroxyapatite and tricalcium phosphate), and that due to their special properties, they are osteoinductive and osteoconductive [1–4].
B2 So far a vast variety of shapes and sizes have been developed, and it’s proved that the most fascinating ones specifically for the purpose of cell delivery, is the ones with spherical form and varying from tens to hundreds of micrometers (i.e., microspheres) in size. The material’s physicochemical properties and sphere morphology highly affects the function of these cells on the microsphere. The supporting substrates and/or containers that hold and deliver cells are called microspheres, and that’s how three-dimensional (3D) micro-environmental cues are provided, and they are successively useful in the development of the tissue [B2].
B1 Micro-carriers (MCs) is widely used in bone tissue engineering. It makes it possible to create a high surface area for the cells. And production of high-yield culture of anchorage- dependent cells is the result . MC’s physio-chemical properties determine how the MCs would be created by the cells. And in this case the micro environments are roughness, chemistry of the surface, and the cell adhesion motifs’ exposure .
B1 As soon as the MCs, under an appropriate condition are loaded with cells, the extracellular matrix (ECM) molecules might be secreted, thus the homogeneous cell construct can be made. These constructs could be added, and injected into the defect zones. A fully interconnected porous construct, which contains an adequate number of viable cells, would be end result . Under many conditions-dynamic or static, cells can be cultured on MCs. There are problematic static conditions, which have low seeding efficiency, and a limited culture medium . In addition, having high concentration gradients and inefficient gas– liquid oxygen transfer are the disadvantages of static cultures . On the other hand, to overcome these disadvantages, dynamic culture conditions are vastly used to seed and proliferate the cells on MCs [5–8]. Spinner flask system, with its gentle agitations is the most common MC culture. It’s proved that for polymeric materials, this technique shows good results, specifically where the MCs and culture medium have almost similar densities , and that’s how, within the culture medium a homogenous suspension of the MC can be created. These conditions improve the gas–liquid oxygen transfer, as a result of that the rate of the survival of the cell and achieving high cell numbers in culture became possible . Due to the fact that polymeric materials lack osteo- conductive and osteo-inductive properties, their capacity is very limited as bone-forming material [B1]. Therefore, for example hydroxyapatite (HA) which is one of the bone-mimicking biomaterials have a vast range of use for bone reengineering [11, 12].
B1 As it is shown in figure 1, this work studies the techniques of using dynamic cell culturing with microporous HA and gelatin/HA-MC as and approach for increasing the output of the cell culture, comparison of their behavior in dynamic cultures as contrasted with their behavior with static ones. For different densities, different behaviors are expected [B1].
Material & Methods and Results:
B1 To produce micro-carriers, aqueous α-tricalcium phosphate, mixed in oil was emulsified. And hydroxyapatite micro-carriers were produced after it has been set. And to produce hybrid gelatin/hydroxyapatite-micro-carriers, it’s enough to add gelatin to the α-tricalcium phosphate. Initially the speed of the dynamic culture caused the attachment of the cells on the micro-carriers, and it was better in low speed (40r/min) in comparison with higher speed (80r/min). Under the average speed of the culture (40 r/min), and in 3 days there was an increase in the number of the cells present in the culture, and specifically in the gelatin micro-carriers. Hydroxyapatite MCs in dynamic culture and for a slightly longer time produced extracellular proteins, which in comparison with a static one contained more cell [B1].
B1 MCs is being used under dynamic cell culture conditions for tissue engineering purposes, and it is one of the most popular methods. This method is also used for cell expansion in comparison with the conventional two- dimensional (2D) culture flask[18, 19]. One of the main nuances was the determination of the optimum conditions could ensure a high rate of the attachment and proliferation of cells. As the most important parameters we can mention; the amount of culture medium , the size and shape of the impeller , the rotation speed[21,22], and the ratio between cell and MCs . Also irregular rotation regimes of the culture can be helpful [1, 23, 24].
B1 Among the parameters already mentioned, rotation speed, greatly influences the proper cell attachment. A higher re-suspension of the MCs in the culture medium might be seen in result of High speeds (80 r/min), however both MC integrity and cell viability were reduced, which are important parameters, therefore, the lower speed (40 r/ min) was chosen. Previously it was mentioned that Micro-Carriers are influenced by the speed [21, 22]. According to previous reports speeds around 40 and 60 r/min give a highest level of proliferation for Vero cells on Cytodex MC, it’s clear that speeds higher or lower than this limit, reduces the quantity of the proliferating cells [25, 26]. At higher speeds of rotation, cells can be damaged by collisions during agitation, and that’s why high speeds must be avoided . As soon as the influence of speed was found out, the dynamic and static conditions were compared in the initial cell seeding on the MCs. In result very prominent and important differences were discovered. Within the first 3 days, there were no significant increase in the number of cells, in the HA-MCs and GEL/HA-MCs in static cultures. On the other side, CTRL-MCs in static conditions shoed a good proliferation, and that’s due to the fact that the surface of the MCs is optimized for anchorage-dependent cells for a better attachment and proliferation to increase in number [B1].
The proliferation of cell on low-temperature calcium phosphates is low, and it’s known that it’s due to surface chemistry and exceeding the roughness of the surface [13, 14, 28–30]. Usually there is a latent delay in proliferation and the quantity of the materials and cells is low at short intervals. Therefore to improve the physio-chemical and biological properties of the CPC, the polymeric additives such as gelatin is added . Therefore, in the long-term and even when the system was subjected to gentle agitation periods, it was noticed that the MC aggregated among them. Within 1st and 2nd weeks the clusters of MC with high amounts of cells were formed. It was a successful result as the surface of the MC was confluence by cells. Controlling different conditions of the culture can help to control the level of aggregation. They are intensity of mixing [32, 33], tension of oxygen , and duration of culture . MC’s suspension efficiency and efficacy is highly affected by their composition. During the stirring periods the CTRL-MCs were easily suspended, which allowed the close interaction of the MCs and cells. As the agitation is over, both precipitated and sedimented, and remained in static for a while, and the MCs and the cells were attached. On the other hand, the MCs containing HA were partially suspended within the primary stages of the culture. Therefore, at each stirring cycle the cells, which were not attached to the MCs, were re-suspended, therefore in comparison to the static culture, their chance to attach to the MCs was highly increased. In result, the denser HA-MCs and GEL/ HA-MCs during the spinner flask culture were partially suspended.
Using dynamic spinner flask culture in comparison to static culture result to a higher rate of cell attachment and proliferation on the HA-containing MCs. The cell attachment and proliferation was improved by adding of gelatin in the HA-MC. After one week of dynamic culture, three-dimensional (3D) tissue engineering constructs were observed which contained MC, cells, and ECM proteins. It is necessary to do more researches in vivo implantation studies to prove the osteogenic potential of the micro-carriers and their future roles.