Trans-septal delivery of hydrogel-encapsulated human umbilical cord MSC-derived neurospheres for acute neuroprotection in traumatic brain injury

APL Bioeng, 06/01/2026

Introduction

The human central nervous system is particularly vulnerable to injury, and brain trauma often results in severe and long-lasting neurological sequelae. Traumatic brain injury (TBI) can lead to motor impairment, cognitive dysfunction, and emotional instability, and even mild TBI may cause structural alterations in the hippocampus and long-term cognitive deficits. Although various therapeutic approaches have been applied, their effectiveness in promoting neural tissue regeneration and functional recovery remains limited. Notably, secondary injury mechanisms occurring over days to weeks after the initial insult further exacerbate neuronal loss, thereby contributing to persistent neurological impairments.

Apoptosis plays an essential role in development and tissue homeostasis; however, its excessive activation is associated with numerous neurodegenerative diseases. Meanwhile, neurogenesis is not restricted to developmental stages but continues in the adult brain, particularly in the hippocampus, and plays a crucial role in learning, memory, and emotional regulation. Following brain injury, an imbalance between programmed cell death and neuroregenerative capacity directly contributes to cognitive and behavioral deficits. Therefore, therapeutic strategies that simultaneously inhibit apoptosis and promote neurogenesis are considered fundamental to neuroprotective and neuroregenerative therapies for TBI.

Given the limited intrinsic regenerative capacity of the central nervous system, stem cell–based therapies have attracted considerable attention as a potential approach for TBI treatment. Preclinical studies have shown that neural stem cells can differentiate into neurons and glial cells after transplantation, thereby contributing to cell replacement and functional recovery. However, limited availability and ethical concerns related to their sources have driven the search for more suitable alternatives. Among these, mesenchymal stem cells (MSCs) derived from bone marrow, adipose tissue, and especially human umbilical cord are considered favorable sources due to their ease of isolation and expansion, as well as their immunomodulatory properties that reduce the risk of immune rejection. Under appropriate induction conditions, MSCs can exhibit neurogenic characteristics; however, their neuroregenerative efficacy and functional recovery after TBI still require improvement through optimization of cell form, survival, and tissue integration following transplantation.

One of the major challenges of cell therapy is the low survival rate of transplanted cells within the injury site, poor retention in brain tissue, and limited differentiation efficiency. Although stimulation of endogenous stem cells may contribute to recovery, the extent of regeneration is often insufficient to compensate for the widespread neuronal loss observed in TBI. Therefore, the development of effective cell delivery systems plays a pivotal role in enhancing therapeutic efficacy. Hydrogel-based biomaterials have been widely applied in neural tissue engineering due to their ability to support cell adhesion, proliferation, migration, and differentiation, while also protecting cells from hostile factors at the injury site and regulating the release of growth factors.

Based on the limitations of current therapies, this study establishes a protocol to generate neurospheres derived from human umbilical cord mesenchymal stem cells and encapsulate them within a silk fibroin/GelGMA hydrogel to enhance cell survival and retention in injured brain tissue. Based on evidence that MSC-derived neurospheres possess neurogenic differentiation potential and superior migratory capacity compared with single-cell suspensions, we hypothesize that neurospheres embedded in a brain-compatible hydrogel may improve neuroprotective efficacy during the acute phase by enhancing cell survival, maintaining paracrine effects, and reducing oxidative stress as well as dendritic degeneration in the hippocampus after injury. To minimize the invasiveness of the treatment, the cell-laden hydrogel system is administered via the trans-septal route, taking advantage of a direct pathway to the brain without the need for surgical intervention.

Results

Phenotype and cellular characterization of hUC-MSCs

• MSCs retained the characteristic spindle-shaped morphology of mesenchymal stem cells after bioreactor culture and cryopreservation.
• Flow cytometry analysis showed that the cells were positive for MSC markers (CD44, CD73, CD90) and negative for hematopoietic lineage markers (CD14, CD19, CD34, CD45), indicating that surface phenotypes were not altered by bioreactor culture conditions.
• MSCs maintained their multilineage differentiation potential into adipogenic, osteogenic, and chondrogenic lineages under appropriate induction conditions.
• These results indicate that large-scale bioreactor-based culture does not compromise the core biological properties of MSCs, supporting their suitability for subsequent applications.

Neurosphere induction of hUC-MSCs

• After thawing, hUC-MSCs were cultured as a monolayer and harvested at passages 5–8 prior to neurosphere induction.
• Cells were transferred to spheroid formation medium in non-adherent plates, began to aggregate after 24 hours, and formed well-defined neurospheres by day 5.
• To induce neurogenic differentiation, neurospheres were cultured in neurodifferentiation medium (containing FBS, L-glutamine, NEAA, N2, B27, and antibiotics) for 7 days.
• Subsequently, neurospheres were transferred back to spheroid formation medium, allowing further maturation with increased size and more stable structure.

Based on previous studies showing that MSC-derived neurospheres can exhibit increased expression of neural progenitor markers (SOX2, Nestin) and enhanced neurogenic potential, the authors refer to these structures as hUC-MSC–derived neurospheres (HMNS). However, since neural markers were not directly evaluated in this study, complete lineage conversion cannot be confirmed.

HMNS attenuate injury in the in vitro TBI-mimicking scratch model

To evaluate the regenerative potential of neurospheres, two complementary in vitro assays were performed using different cell lines to match specific evaluation endpoints. The hippocampus-derived HT22 cell line was used to assess cell viability, as it is relevant to neuronal injury in TBI models, whereas the highly migratory Neuro-2a cell line was selected to evaluate cell migration and wound closure capacity.

Mechanical injury induced by the scratch assay significantly reduced the viability of HT22 cells. In contrast, treatment with neurosphere-loaded hydrogel at doses of 100 and 200 clusters markedly improved cell survival compared with the hydrogel-only group.

Although the difference between the two doses did not reach statistical significance, the 200-neurosphere group showed a more consistent trend toward improvement and was therefore selected as the representative dose for subsequent in vivo experiments.

In the wound-healing assay, Neuro-2a cells were co-cultured under the following conditions: no scratch, GelGMA/SF hydrogel alone, and hydrogel containing HMNS at doses of 100 or 200 neurospheres. Samples were monitored at 0, 12, and 24 hours. The results demonstrated that neurosphere-loaded hydrogels significantly promoted cell migration, with the 200-neurosphere group achieving the highest wound closure rate after 24 hours of culture.

Trans-septal transplantation of hydrogel-encapsulated HMNS exerts neuroprotective effects and improves neurological function after TBI

• Evaluations were performed in a mouse TBI model with longitudinal behavioral (mNSS, ΔmNSS) and histological assessments.
• The group treated with hydrogel-encapsulated HMNS delivered via the intranasal route showed decreased mNSS and increased ΔmNSS, indicating better neurological functional recovery compared with controls.
• Histological analysis at 7 days post-injury revealed:

+ BDNF levels in the CA1 region were markedly reduced after TBI but were clearly restored in the HMNS-treated group.

+ An increased number of NeuN-positive neurons in the CA1 region, the dentate gyrus granule cell layer, and the hilus.

• These findings demonstrate the neuroprotective effects of HMNS and enhanced neuronal survival in the hippocampus.

Trans-septal transplantation of HMNS reduces nitrosative stress, oxidative stress, and dendritic cell loss after TBI

• Brain tissues were collected 7 days after TBI and stained with:

+ Fluoro-Jade B (FJB) → neuronal degeneration.

+ Nitrotyrosine → nitrosative stress.

+ 4-hydroxynonenal (4-HNE) → oxidative stress.

+ Microtubule-Associated Protein 2 (MAP2) → dendritic integrity.

• In the TBI-only group:

+ A marked increase in FJB-positive neurons in the granule cell layer (GCL) and hilus.

+ Elevated nitrotyrosine and 4-HNE levels.

+ Reduced MAP2 expression in the CA1 region and dentate gyrus (DG).

• In the group treated with hydrogel-encapsulated HMNS:

+ A significant reduction in neuronal degeneration.

+ Decreased oxidative and nitrosative stress.

+ Preservation of dendritic structure.

These results demonstrate the multimodal neuroprotective effects of HMNS following TBI.

Discussion

Traumatic brain injury (TBI) induces prolonged inflammatory responses and progressive neuronal degeneration, creating a hostile microenvironment for tissue repair and increasing the risk of chronic neurological disorders. TBI not only leads to motor, cognitive, and emotional impairments, but there is currently no specific pharmacological therapy that effectively promotes neural tissue regeneration and functional recovery.

In this context, stem cell–based therapy, particularly using mesenchymal stem cells (MSCs), is considered a promising strategy due to their immunomodulatory and neuroprotective properties. MSCs can attenuate inflammation, secrete neurotrophic factors that enhance neuronal survival, inhibit apoptosis, and promote neural tissue regeneration. In addition, culturing umbilical cord–derived MSCs in the form of neurospheres enhances their neurogenic characteristics and migratory capacity.

Intranasal (trans-septal) delivery represents a minimally invasive route that can bypass the blood–brain barrier via the olfactory and trigeminal nerve pathways. Encapsulation of neurospheres within hydrogels improves cell survival, enhances retention at the target site, and increases therapeutic efficacy. Therefore, this study evaluated the therapeutic effects of hydrogel-encapsulated HMNS delivered via the trans-septal route for the treatment of TBI.

The results demonstrated that HMNS improved cell survival and migration in vitro and promoted neurological functional recovery in an animal TBI model. At the histological level, HMNS increased BDNF expression, enhanced neuronal survival, and reduced neuronal degeneration, oxidative/nitrosative stress, and dendritic damage, indicating both neuroprotective and neurorestorative effects. Although limitations remain regarding control groups and follow-up duration, these data support intranasal delivery of hydrogel-encapsulated HMNS as a feasible and minimally invasive therapeutic strategy for TBI.

Conclusion

This study demonstrates that neurospheres derived from human umbilical cord mesenchymal stem cells (hUC-MSCs), encapsulated in silk fibroin/GelGMA hydrogel and delivered via the trans-septal intranasal route, exhibit significant therapeutic potential for traumatic brain injury (TBI). In vitro, HMNS enhanced cell survival and migration; in an animal TBI model, HMNS improved neurological function, restored hippocampal BDNF expression, preserved neurons, reduced oxidative and nitrosative stress, and attenuated dendritic damage. These findings indicate that HMNS can modulate the post-injury microenvironment and exert early neuroprotective effects, thereby contributing to improved functional recovery. Overall, hydrogel-encapsulated HMNS represents a promising regenerative therapeutic strategy for TBI, and future studies should focus on optimizing delivery methods, elucidating underlying mechanisms, and advancing clinical translation.

References

Dong Wook Kim, Ok Joo Lee, Bo Young Choi, Md. Tipu Sultan, Olatunji Ajiteru, Min Kyu Park, Ji Seung Lee, Soon Hee Kim, Kyu Young Choi, Sang Won Suh, Chan Hum Park; Trans-septal delivery of hydrogel-encapsulated human umbilical cord MSC-derived neurospheres for acute neuroprotection in traumatic brain injury. APL Bioeng. 1 March 2026; 10 (1): 016103.

Source: APL Bioeng

Link: https://pubs.aip.org/aip/apb/article/10/1/016103/3376451