Transplantation of SCs at the site of nerve transection has been previously shown to promote peripheral nerve regeneration, but these studies have not evaluated the ability of syringe injection to successfully transfer SCs. In this study, we systematically evaluated the transplantation of SCs into acellular CP nerve grafts through syringe injection. The current study (1) determined the needle gauge for maximal viability of SCs during injection into nerve grafts, (2) tracked the seeded SCs in vitro, and (3) evaluated the efficiency with which SCs are seeded within acellular nerve grafts using syringe injection.
Needle size is a critical parameter for cell transplantation by injection into nerve grafts. The inner diameter of the needle must be large enough to avoid excessive shear force on the cells, and the outer diameter of the needle must be small enough to avoid tearing of the epineurium. A standard Live/Dead assay was performed to determine the needle gauge would provide the maximal number of viable SCs (after the cells are expelled from the needle). A significant increase in viability was observed for the 24, 27, and 29-gauge needles compared to the 33-gauge needle. The inner diameters of the 24, 27, and 29-gauge needles were large enough to allow the SCs to prevent significant shear forces on the cell membrane. The rate at which the cells are expelled from the needle may introduce mechanical stress, which may compromise the integrity of the cell membrane and eventually lead to cell death (McNeil, 1993
). In contrast, the smaller inner diameter of the 33-gauge needle adversely affected the survival of the SCs by likely subjecting the injected cells to intolerable levels of shear force. The outer diameter of the injection needle must also be considered to safeguard against undue damage to the epineurium during the seeding process. Damage to the epineurium would cause the newly seeded SCs to leak from the grafts after injection. Injection with the 24-gauge needle caused damage to the epineurium, whereas the 27-gauge needle allowed successful SC seeding without noticeable damage to the epineurium. Given the combined effects on viability and epineurium integrity, it was determined that the 27-gauge needle was the best option for injecting SCs into acellular nerve grafts.
Injecting under the epineurium does not guarantee successful transfer and seeding of SCs. Developing a method to determine that the injection was successful prior to graft implantation would decrease the probability of experimental error due to low SC numbers within the graft. Previous work in this field failed to track seeded SCs prior to graft implantation (Fox et al., 2005a
; Fox et al., 2005b
). The inability to monitor injection success could result in implantation of grafts that lack SCs due to injection error and thus contribute to experimental error.
Commercially available fluorescent dyes can be used to label and track cells following injection. One such dye shown to be effective in SCs is the 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). The advantage of labeling SCs with CFSE is that the transplanted SCs can be distinguished from the host SCs over long periods after transplantation (up to 4 weeks in some studies) due to the tolerance of SCs to CFSE (Li et al., 2003
). An alternative method involves the Qtracker® dye (Invitrogen), which uses Qdot nanocrystals to label and track transplanted cells (Chakraborty et al., 2007
; Jaiswal et al., 2003
; Riegler et al., 2008
). In the current study, the Qtracker® was chosen over the CFSE because of the ease of labeling cells: Q-tracker® has been shown to provide an intense fluorescence for cells, can be traced through cell division cycles, and is not transferred to adjacent cells. Studies have used this dye in cell motility, migration, and a number of other functional assays. One distinct feature of Q-tracker® is that it uses a custom targeting peptide to deliver Qdot nanocrystals into the cytoplasm of cells. It is therefore easily adaptable for other cell types (Futaki, 2005
; Wender et al., 2002
). Also, labeling with Qtracker® requires only a simple incubation for one hour, after which the cells are stable for injection for at least 2 hours, giving us a more flexible time frame for our experiments. In contrast, labeling with CFSE requires incubation with the dye in combination with other chemicals, and the cells must be used immediately after labeling due to instability of the dye.
Qtracker® allowed for qualitative assessment of SCs injection into the graft by fluorescence microscopy. Grafts injected with106 SCs exhibited a higher fluorescence intensity compared to those injected with 105 SCs which exhibited little to no fluorescence. The Qtracker® dye provided the ability to track seeded SCs in CP acellular nerve grafts, which can provide quality control prior to experimental graft implantation in future studies.
Another important factor that affects success or failure of cell transplant therapies is the efficiency with which the therapeutic cells are transferred into the desired tissue. Stereological analysis of grafts after injection revealed that only a fraction of the injected SCs remained in the graft. The failure to successfully transfer all of the cells may be either due to the death of cells post injection, the loss of SCs leaking from grafts post-injection and prior to fixation or the staining efficiency of Qtracker ®. Only 10% of the cells were successfully transferred when 106
SCs were injected into the CP acellular nerve grafts and only 40% were successfully transferred when 105
SCs were injected. Previous studies with CP nerve grafts showed that injection with 105
SCs had little effect on peripheral nerve regeneration (Fox et al., 2005b
), while injection with 106
SCs enhanced regeneration over CP grafts alone (Brenner et al., 2005
). Based on the current work, we hypothesize that seeding efficiency of cells transferred may have played an important role in the lack of therapeutic outcomes with respect to nerve regeneration in our previous studies. Additionally, the combined results of the current study and our previous studies suggests that there may be a threshold number of SCs necessary to elicit a therapeutic effect in acellular nerve grafts. Mechanical stress may also limit the number of viable cells transferred to the grafts. To reduce the stress on the cells, the injection rate can be optimized to deliver the maximum amount of viable cells to the graft post injection. The number of cells transferred to the grafts after incubation in growth media is much lower than the desired 106
cells, which may have allowed the cells to leak into the media from the graft because they could not successfully adhere to the graft or cell death resulting from insufficient perfusion of the graft in the cell culture media. The staining efficiency (~70% in this study) of the Qtracker can also be determined and increased for future studies to ensure that a sufficient number of cells are labeled prior to injection. To maximize the number of cells transferred to the graft, it is beneficial to use the grafts to repair the injury immediately after the cells are injected, as the number of viable cells decreased with time (). A combination of these strategies may increase the seeding efficiency of cells, which may further enhance the effects of transplanted SCs on peripheral nerve regeneration.
From a clinical perspective, we need to consider the source of the SCs used for transplantation as well as the purity of the SCs. The SCs can be derived from the transected nerve itself. A piece of the nerve from the stump can be removed and the SCs can be harvested and cultured as described in the methods. The cell population can be purified by using selection techniques such as immunopanning (Barres et al., 1992
) or Dynal magnetic beads (Invitrogen) (Neurauter et al., 2007
). SCs express the surface receptor p75NTR
(Lemke and Chao, 1988
), so SCs can be bound to an anti-p75NTR
antibody coated on a Petri dish for immunopanning or on magnetic beads to help purify a pure population of SCs. It is critical to transplant a pure population into a patient to ensure that the transplanted cells will help the injured nerve to regenerate. Our goal in this study was to develop a systematic method to evaluate and determine how to inject cells into acellular grafts for transplantation therapies. We have shown that using these evaluation methods that we are able to successfully re-introduce cells into acellular grafts, which is important to ensure the success of in vivo